TNF receptor I is required for induction of macrophage heat shock protein 70

Julie K. Heimbach1, Leonid L. Reznikov2, Casey M. Calkins1, Thomas N. Robinson1, Charles A. Dinarello2, Alden H. Harken1, and Xianzhong Meng1

Departments of 1 Surgery and 2 Medicine, University of Colorado Health Sciences Center, Denver, Colorado, 80262


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Expression of heat shock proteins (HSP) is an adaptive response to cellular stress. Stress induces tumor necrosis factor (TNF)-alpha production. In turn, TNF-alpha induces HSP70 expression. However, osmotic stress or ultraviolet radiation activates TNF-alpha receptor I (TNFR-I) in the absence of TNF-alpha . We postulated that TNF-alpha receptors are involved in the induction of HSP70 by cellular stress. Peritoneal Mphi were isolated from wild-type (WT), TNF-alpha knockout (KO), and TNFR (I or II) KO mice. Cells were cultured overnight and then heat stressed at 43 ± 0.5°C for 30 min followed by a 4-h recovery at 37°C. Cellular HSP70 expression was induced by heat stress or exposure to endotoxin [lipopolysaccharide (LPS)] as determined by immunoblotting. HSP70 expression induced by either heat or LPS was markedly decreased in TNFR-I KO Mphi , whereas TNFR-II KO Mphi exhibited HSP70 expression comparable to that in WT mice. Expression of HSP70 after heat stress in TNF-alpha KO Mphi was also similar to that in WT mice, suggesting that induction of HSP70 by TNFR-I occurs independently of TNF-alpha . In addition, levels of steady-state HSP70 mRNA were similar by RT-PCR in WT and TNFR-I KO Mphi despite differences in protein expression. Furthermore, the effect of TNFR-I appears to be cell specific, since HSP70 expression in splenocytes isolated from TNFR-I KO was similar to that in WT splenocytes. These studies demonstrate that TNFR-I is required for the synthesis of HSP70 in stressed Mphi by a TNF-independent mechanism and support an intracellular role for TNFR-I.

gene knockout; tumor necrosis factor-alpha ; reverse transcription-polymerase chain reaction; splenocyte


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EXPRESSION OF HEAT SHOCK PROTEINS (HSPs) is an endogenous mechanism promoting cellular adaptation to stress. The 70-kDa HSP family includes the inducible HSP72 and the constitutive HSP73. Though the functions of this family of stress proteins remain incompletely defined, the HSP70 family functions in both stress and nonstress conditions as a chaperone molecule that directs protein translocation within the cell, the folding of de novo proteins, and the degradation of denatured proteins (5, 15).

Induction of HSP70 confers increased survival following cellular insult and occurs after exposure to a variety of stressors including heat shock (12, 16), hypoxia (6), hemodynamic stress (13, 17), ultraviolet (UV) radiation (27), and oxidant stress (26). Recent studies also suggest that HSP70 is induced after exposure to proinflammatory factors such as endotoxin [lipopolysaccharide (LPS)] or tumor necrosis factor (TNF)-alpha (21). In addition, HSP70 expression inhibits proinflammatory cytokine production by macrophages (25).

TNF-alpha is a proinflammatory cytokine that binds two distinct members of the TNF-alpha receptor superfamily. Activation of the p55 TNF-alpha Receptor I (TNFR-I) is responsible for many well-characterized effects of TNF-alpha including translocation of nuclear factor (NF)-kappa B (7, 23), activation of the inflammatory cascade (4), and initiation of apoptosis (28, 32). In addition to triggering a proinflammatory cascade, however, TNF-alpha signals a compensatory cytoprotective cellular response. This self-preservational profile is characterized by the production of anti-inflammatory cytokines such as interleukin (IL)-10 (31), production of IL-1 receptor antagonist (30), release of soluble cytokine receptors such as soluble TNFR-I and TNFR-II (29), and synthesis of cellular HSP70 (21). Targeted activation of this endogenous cytoprotective cascade is an appealing clinical strategy to combat the systemic inflammation that occurs in patients after severe injury.

Macrophages are often the source of the key mediators fueling the posttraumatic systemic inflammatory response and, thus, should be an ideal target for a therapeutic anti-inflammatory strategy. The role of TNF-alpha in macrophage HSP70 induction and the role for heat shock in TNFR-I activation have not been described. Notably, however, UV light and hyperosmotic stresses directly activate TNFR-I in HeLa cells (19). Rosette and Karin (19) report TNFR-I clustering and recruitment of the TNFR-I-associated death domain (TRADD) after exposure to UV radiation or hyperosmotic stress. Investigation of the role of the TNF-alpha receptor in heat stress-induced HSP70 expression is important for exploring the mechanism by which cells sense a stress as well as in understanding the pathways responsible for the dynamic proinflammatory/cytoprotective balance within the cell.

Given the observation that the TNFR-I can be directly activated by physical stress in the absence of its ligand (19), and given the simultaneous pro- and anti-inflammatory responses initiated by TNF-alpha , we proposed a relationship between TNFR-I and the induction of HSP70. Therefore, the purposes of this study were to 1) determine if TNFR-I and/or TNFR-II is required for induction of HSP70 in Mphi following heat stress or LPS, 2) evaluate whether TNF-alpha is required for induction of HSP70 by heat stress, 3) ascertain whether the role of TNFR-I or TNFR-II in HSP70 induction is cell specific, and 4) determine whether TNF receptors regulate HSP70 synthesis at the transcriptional or posttranscriptional level.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Reagents. Dulbecco's modified Eagle's culture medium (DMEM/F-12) supplemented with 10 mM L-glutamine, 24 mM NaHCO3, 10 mM HEPES, 10% fetal calf serum (FCS), and 150 ug/ml gentamicin were purchased from GIBCO BRL (Gaithersburg, MD). FCS was purchased from Life Technologies (Grand Island, NY). Lyophilized LPS (a phenol-extracted preparation from Escherichia coli 055:B5) as well as other reagents unless otherwise noted were purchased from Sigma Chemical (St. Louis, MO). Murine TNF-alpha was obtained from R&D Systems (Minneapolis, MN). Four-domain soluble TNFR-I binding protein (TNF bp) was a gift of Dr. Carl Edwards (Amgen, Thousand Oaks, CA). The polyclonal rabbit antibody against the inducible form of HSP70 was purchased from StressGen Biotechnologies (Victoria, BC, Canada.) This antibody has no cross-reactivity to the constitutive isoforms of the HSP70 family.

Mice. Mice deficient in the TNF p55 receptor [TNFR-I knockout (KO)], deficient in the TNF p75 receptor (TNFR-II KO), and matching the C57BL/6J wild-type background were obtained from Jackson Laboratory (West Grove, PA). TNF-alpha KO mice were generous gifts from Dr. D. W. Riches (National Jewish Medical Center, Denver, CO). Matching wild-type animals (B6-129) for TNF-alpha KO were obtained from Jackson Laboratory. Animals were 7-9 wk old at the time of study and were housed at the University of Colorado Health Sciences animal care facility for at least 1 wk before being studied. Animals were allowed free access to food and water and were exposed to 12:12-h light-dark cycles before death. Animal experiments were approved by the Animal Care and Research Committee, University of Colorado Health Sciences Center. Animals received care in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892]. Animals were killed with 50 mg/kg pentobarbital sodium by injection.

Cell culture and treatment. Peritoneal Mphi were isolated by peritoneal lavage. The peritoneal cavity was lavaged twice (7 ml each) with filter-sterilized ice-cold DMEM/F-12 and kept at 4°C. Cells pooled from animals of the same genotype were centrifuged at 500 g for 10 min at 4°C and then washed once with DMEM/F-12. Cells were counted with a hemacytometer and cultured at a density of 1-2 × 106 cells/ml in 20-mm tissue culture plates at 37°C in a humidified 5% CO2 atmosphere. Nonadherent cells were removed after 4 h at 37°C by washing once with warm medium. The adherent cells were allowed to recover overnight. Cell viability was confirmed at >95% using trypan blue exclusion. Cells were treated with heat stress at 43.0 ± 0.5°C for 30 min. For HSP70 protein assays, cells were allowed a 1-h recovery at 37°C before cells were collected. For RNA isolation, cells from a parallel experiment were collected after a 15-min recovery period at 37°C. Cells were collected with a rubber scraper and gentle medium washes repeated three times and were then stored as a dry pellet at -70°C.

Splenocytes were harvested as previously described (18). Briefly, after aseptic removal of spleens, cell suspensions in RPMI with 10% fetal bovine serum were prepared as previously described (2). Spleen cells were cultured in 1.0 ml at 5 × 106 cells/ml in 24-well, flat-bottom culture plates in the presence or absence of LPS (10 µg/ml). Cultures were incubated at 37°C in a humidified 5% CO2 atmosphere. For treatment with LPS or TNF-alpha , cells were cultured overnight as described and then exposed to either medium containing 500 ng/ml E. coli LPS or 15 ng/ml TNF-alpha for 4 h. Cells were washed and pelleted for protein extraction. For experimental conditions requiring pretreatment with TNFR-I binding protein, 10 ng/ml TNF bp was applied 30 min before LPS treatment.

Immunoblotting. Cell density was adjusted to a final concentration of 1 × 107 cells/ml in a lysis buffer containing 25 mM Tris · HCl, 2% SDS, 0.02% bromphenol blue, and 10% glycerol at pH 6.8. Samples were boiled for 5 min, vortexed, and placed on ice before loading. Protein concentration was measured by Coomassie Plus protein assay using BSA as the standard (Pierce, Rockford, IL). Electrophoresis was performed using 4-20% linear gradient SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). After electrophoretic transfer to nitrocellulose membranes (Bio-Rad), membranes were stained with Ponceau S solution (Sigma) to assess equality of protein loading. After densitometry, membranes were washed and incubated in PBS containing 5% milk for 1 h. Membranes were then incubated in PBS with 0.1% Tween 20, 5% milk, and a 1:5,000 dilution of polyclonal antibody against HSP70 (StressGen). Horseradish peroxidase-conjugated goat anti-rabbit IgG at a 1:5,000 dilution was used as a detection antibody. The bands were visualized using an ECL (enhanced chemiluminescence) kit (Pierce) for 1 min, followed by exposure to film. Quantification of the immunoblots was performed by computer-assisted densitometry (NIH application 1.599b4). Relative density values are expressed as a percentage of the control value for each murine genotype evaluated. All densities are reported as means ± SE.

Murine cytokine assays. TNF-alpha in cell supernatants was determined using a murine TNF-alpha ELISA kit (R&D Systems) with a detection limit of 13.5 pg/ml. Samples were run undiluted, in duplicate.

RNA isolation and RT-PCR. Total RNA was isolated from peritoneal macrophages or splenocytes using Tri-Reagent (Molecular Research Center, Cincinnati, OH). Cells were lysed in Tri-reagent, and RNA was sequentially isolated using chloroform extraction and isopropanol precipitation. RNA was dissolved in diethyl pyrocarbonate-treated water and quantified using GeneQuant (Pharmacia Biotech, Cambridge, UK). To prepare cDNA, 0.5-2 ug of total RNA were reverse-transcribed using random primers in a final concentration of 5 mM MgCl2, 50 mM KCl, and 10 mM Tris · HCl (pH 8.3), 1 mM of each dNTP, 20 units of RNase inhibitor, and 50 units of MuLV reverse transcriptase (Perkin Elmer, Branchburg, NJ.) The reaction was incubated at 42°C for 40 min and terminated at 95°C for 5 min. PCR was performed using HotStarTaq DNA polymerase and the Q-Solution Kit (Qiagen, Valencia, CA) in accordance with the manufacturer's protocol. Briefly, 2 µl of RT product were used in a total volume of 50 µl containing 5 µl of 10× PCR buffer, 10 µl of 5× Q-solution, 0.2 mM of each dNTP, and 1.5 units of HotStarTaq Polymerase (Qiagen). The sense primer for HSP70 was 5'-AAACTCCCTCCCTGGTCTGA, and the reverse primer was 5'-CTTGTCTTCGCTTGTCTCTG. The sense primer for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was 5'-ACCACAGTCCATGCCATCAC, and the reverse primer was 5'-AGGTGGTGGGACAACGACAT (GIBCO BRL, Rockville, MD). PCR was performed on a Peltier Thermal Cycler-200 (MJ Research, Watertown, MA). For each PCR reaction, the following sequence was used: preheating at 95°C for 15 min, then at 94°C for 40 s, 55°C for 45 s, and 75°C for 1 min, with a final extension phase at 72°C for 10 min. A variable number of cycles from 25 to 40 were used to ensure that amplification occurred in the linear phase and that differences between control and experimental conditions were maintained by adopting a limited number of cycles. The optimal number of cycles was established at 35. The PCR amplification using GAPDH as the internal control was performed on each sample to ensure that differences between tubes were not the result of unequal concentrations of RNA. The PCR products were separated on a 1.5% agarose gel containing 0.5× TBE (50 mM Tris, 45 mM boric acid, 0.5 mM EDTA, pH 8.3) with 0.5 ug/ml ethidium bromide, visualized by UV illumination, and photographed. Densitometry was performed on the negative image (ImageQuant software; Molecular Dynamics, Sunnyvale, CA), and the relative absorbance of the HSP70 PCR products was corrected against the absorbance obtained for GAPDH.

Statistical analysis. Data were expressed as means ± SE. An analysis of variance was performed, and a difference was accepted as significant with P < 0.05 verified by a Bonferroni/Dunn post hoc test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of TNF receptors in heat stress induction of Mphi HSP70. Baseline HSP70 levels were undetectable or barely detectable in peritoneal Mphi isolated from wild-type mice and mutant mice lacking TNFR-I or TNFR-II. Heat stress induced HSP70 in peritoneal Mphi isolated from wild-type mice. However, the presence of HSP70 was markedly reduced in the TNFR-I KO cells (Fig. 1). In contrast, stress-induced HSP70 synthesis was preserved in Mphi isolated from TNFR-II KO mice.


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Fig. 1.   A: heat shock protein 70 (HSP70) Western blot analysis of peritoneal macrophages from wild-type (WT), tumor necrosis factor (TNF)-alpha receptor I knockout (TNFR-I KO), or TNFR-II KO mice. Mice were either untreated (control) or subjected to heat stress and recovery as described in MATERIALS AND METHODS. Immunoblot is representative of 4 separate experiments using pooled macrophages from 3-5 animals. B: densitometric analysis of immunoblots from 4 experiments. Band density is reported as the relative increase following heat stress compared with control of same murine genotype. *P < 0.05 vs. control of same genotype; dagger P < 0.05 vs. WT heat stress (by ANOVA).

Role of TNF-alpha in heat stress induction of Mphi HSP70. To determine whether the attenuation of heat stress-induced HSP70 production in TNFR-I KO Mphi was mediated through TNF-alpha , we examined whether heat stress upregulates TNF-alpha release. As a positive control, TNF-alpha was induced by LPS stimulation in Mphi isolated from wild-type mice and mutant mice lacking TNFR-I or TNFR-II. Cells were exposed to media containing 500 ng/ml E. coli LPS for 4 h, and the supernatants were collected. For heat stress treatment, cells were placed at 43°C for 30 min as described in MATERIALS AND METHODS. Supernatants were collected after a 4-h recovery at 37°C. TNF-alpha release was not detected in supernatants following heat stress regardless of the phenotype of the cells (Fig. 2). Given the described autocrine function of TNF-alpha (24), we determined the role of this cytokine in heat stress-induced Mphi HSP70 production using Mphi lacking TNF-alpha . Indeed, HSP70 production was preserved in Mphi isolated from TNF-alpha KO mice (Fig. 3).


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Fig. 2.   TNF-alpha production from peritoneal macrophages. Cells from C57BL6 (control), TNFR-I KO, or TNFR-II KO mice were untreated (baseline), treated with lipopolysaccharide (LPS), or treated with heat stress (h.s.) as described in MATERIALS AND METHODS. Supernatants were collected after 4 h of exposure to LPS or heat. TNF-alpha production was assayed from supernatants using ELISA. LPS was used as a positive control for TNF-alpha production and led to an increase in TNF-alpha in all groups studied. Heat stress caused no increase in TNF-alpha production domains. Experiments were performed using pooled macrophages from 5 animals and were repeated twice. Data are means ± SE. *P < 0.05 vs. baseline and heat stress (by ANOVA).



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Fig. 3.   A: HSP70 Western blot analysis of peritoneal macrophages from WT or TNF-alpha KO mice. Mphi were either untreated (control) or subjected to heat stress as described in MATERIALS AND METHODS. Experiments were performed using pooled macrophages from 3-5 animals. Immunoblot is representative of 3 separate experiments. B: densitometric analysis of immunoblots from 3 experiments. Band density is reported as the relative increase following heat stress compared with control of same murine genotype. *P < 0.05 vs. control of same genotype (by ANOVA).

Role of TNF receptors and TNF-alpha binding protein in induction of Mphi HSP70 by LPS. As in heat stressed Mphi , baseline HSP70 levels were undetectable or barely detectable in peritoneal Mphi isolated from wild-type mice and mutant mice lacking TNFR-I or TNFR-II. However, LPS treatment induced HSP70 in peritoneal Mphi isolated from wild-type mice. As above, HSP70 production was markedly reduced in the TNFR-I KO cells (Fig. 4). However, since LPS treatment induced TNF-alpha production whereas heat stress did not (Fig. 2), the ligand responsible for activation of TNFR-I following these two stressors may be different. To block activation of TNFR-I by LPS-induced TNF production, wild-type Mphi were stimulated with LPS in the presence of saturating concentrations of TNF bp. Treatment of wild-type Mphi with TNF bp before exposure to LPS prevented HSP70 induction (Fig. 5), whereas HSP70 induction by heat stress was preserved (Fig. 6). These data suggest that LPS induction of HSP70 is dependent on activation of TNF receptors by TNF, whereas heat stress induction of HSP70 regulated by TNFR-I is TNF-alpha independent.


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Fig. 4.   A: HSP70 Western blot analysis of peritoneal macrophages from WT or TNFR-I KO mice. Cells were either untreated (control) or subjected to LPS, and after treatment for 4 h, immunoblotting was performed. Experiments were performed using pooled macrophages from 3-5 animals. Immunoblot is representative of 3 separate experiments. B: densitometric analysis of immunoblots from 3 experiments. Band density is reported as the relative increase following LPS compared with control of same murine genotype. *P < 0.05 vs. WT control; dagger P < 0.05 vs. WT LPS (by ANOVA).



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Fig. 5.   A: HSP70 Western blot analysis of peritoneal macrophages from WT mice following LPS treatment. Cells were stimulated with LPS in the presence or absence of soluble TNFR-I binding protein (TNF bp; 10 mg/ml). Experiments were performed using pooled macrophages from 5 animals and repeated twice. B: densitometric analysis of immunoblots from 3 experiments demonstrates that TNF bp prevented HSP70 protein expression in the presence of LPS. Band density is reported as the relative decrease following TNF bp compared with LPS-treated control of the same murine genotype. *P < 0.05 (by ANOVA).



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Fig. 6.   HSP70 Western blot analysis of peritoneal macrophages from WT mice. Cells were treated with heat stress in the absence or presence of TNF bp. Cells were subjected to heat stress and then harvested after a 4-h recovery. Experiments were performed using pooled macrophages from 5 animals and repeated twice. Densitometric analysis of these immunoblots failed to recognize a difference in band density between groups.

Influence of TNFR-I on HSP70 gene expression. We next determined whether the lack of HSP70 production in peritoneal Mphi of TNFR-I KO mice correlated to HSP70 gene transcription. HSP70 mRNA levels were examined using semi-quantitative RT-PCR. HSP70 mRNA was not present in either wild-type or TNFR-I KO cells before heat stress. Heat stress strongly induced HSP70 mRNA production in wild-type cells. Despite the reduced HSP70 protein synthesis in Mphi lacking TNFR-I, HSP70 mRNA levels following heat stress in this phenotype were similar to those in wild-type Mphi (Fig. 7).


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Fig. 7.   Steady-state mRNA levels of HSP70 in peritoneal macrophages. Cells from WT or TNFR-I KO were untreated (cntl) or treated with heat stress (h.s.). mRNA for HSP70 was determined by semi-quantitative PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed to ensure equal RNA loading. Immunoblot is representative of 2 similar experiments. HSP70 mRNA production is not different between WT and TNFR-I KO groups.

Cell-type specificity of regulating HSP70 production by TNFR-I. To investigate whether the influence of TNFR-I on HSP70 production was specific to Mphi , we examined HSP70 production in splenocytes following an exposure to identical heat stress. In contrast to peritoneal Mphi , splenocytes lacking TNFR-I retained the capability of expressing HSP70 mRNA and protein (Figs. 8 and 9).


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Fig. 8.   A: HSP70 Western blot analysis of splenocytes from C57BL6 (WT) or TNFR-I mice. Cells were either untreated (control) or subjected to heat stress and recovery. After 4 h at 37°C, immunoblotting was performed. Immunoblot is representative of 3 separate experiments. B: densitometric analysis of immunoblots from 3 experiments demonstrates that TNF bp prevented HSP70 protein expression in the presence of LPS. Band density is reported as the relative decrease following TNF bp compared with LPS-treated control of the same murine genotype. *P < 0.05 vs. control of same genotype (by ANOVA).



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Fig. 9.   Steady-state mRNA levels of HSP70 in splenocytes. Splenocytes from WT or TNFR-I KO were untreated (control) or treated with heat stress. After a recovery period at 37°C, total cellular RNA was harvested for RT-PCR. Results are representative of 3 separate experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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In the present study, we determined that synthesis of HSP70 is nearly absent in peritoneal Mphi from mice lacking TNFR-I by a TNF-alpha -independent mechanism. However, TNF-alpha has been demonstrated to induce HSP70 in fibroblasts (21). The ability of TNF-alpha to induce HSP70 is thought to be part of the stress response, since direct activation of the TNFR-I by physical stress (UV radiation or osmotic stress) was reported in a previous study (19). In addition, in HeLa cells transfected with antisense TNF-alpha mRNA to inhibit endogenous TNF-alpha , decreased HSP70 production, increased sensitivity to heat, and increased binding of heat shock factor 1 (HSF1) to heat shock element were observed (33, 34). These results suggest that TNFR-I regulates HSP70 expression induced by both TNF-alpha -dependent (LPS) and -independent (heat stress) mechanisms. The regulation is specific to macrophages and appears to be at the level of translation.

In the present study, we have demonstrated a link between TNFR-I and HSP70 expression in the macrophage, a principal cell mediating the inflammatory response. The results of the present study indicate that 1) TNFR-I is required for synthesis of Mphi HSP70 by heat stress; 2) the influence of TNFR-I on heat stress induction of Mphi HSP70 is independent of TNF-alpha , whereas LPS induction of HSP70 depends on TNF-alpha ; 3) the requirement for TNFR-I in HSP70 induction is cell specific, being essential in Mphi but not in splenocytes; and 4) TNFR-I regulates Mphi HSP70 production at the posttranscriptional level.

A relationship between a non-TNF-alpha -activated pathway for TNFR-I and induction of HSP70 has not been previously described. Our data suggest an intracellular role for TNFR-I in the production of HSP70. TNFR-I signaling involves a duality in which pathways leading to apoptosis are activated along with mechanisms of cellular protection. TNFR-I activation leads to trimerization and recruitment of TRADD, a secondary intracellular mediator that subsequently ligates TNF-alpha receptor-associated factor 2 (TRAF2) and Fas-associated death domain (FADD). TRAF2 activates NFkappa -B, which has antiapoptotic effects, while FADD binds both to the proapoptotic cell-surface receptor Fas and is required for TNFR I signaling of apoptosis (8). Activation of Fas inhibits HSF1 and the subsequent induction of HSP70 (22). Because HSP70 has been shown to be antiapoptotic (3, 11), suppression of HSP70 by expression by Fas signaling may ensure the execution of programmed cell death. In the present study, HSP70 production was nearly absent following heat stress or LPS exposure in TNFR-I KO Mphi , suggesting that TNFR-I is required for Mphi HSP70 induction by either stress. Alternatively, the intracellular presence of TNFR-I, rather than its extracellular activation, may be responsible for Mphi HSP70 synthesis. Because TNFR-I activation was not measured in this study, it remains unclear whether TNFR-I influences HSP70 synthesis via activation or by a mechanism independent of receptor activation. However, blocking TNF receptors with TNF bp had an effect on LPS-induced HSP70 induction similar to that in the absence of TNFR-I, suggesting that ligand binding and activation of the receptor plays a role in synthesis of HSP70.

It is always possible that the lack of heat stress induction of HSP70 in the TNFR I KO Mphi is due to a genetic alteration related to a life-long absence of the TNFR-I. However, the ability of TNFR I KO splenocytes to produce HSP70 in response to heat stress argues against an inability to produce HSP70 secondary to altered genetic component from conception. Therefore, despite the limitations of the KO model, the evidence appears compelling in support of a role of the TNFR I in regulating heat stress induction of macrophage HSP70. Posttranslational stability of HSP70 protein may be a factor determining cellular HSP70 level following stress, and intracellular TNFR-I may stabilize HSP70. However, it is unlikely that HSP70 is rapidly degraded in cells lacking TNFR-I, since a similar difference in stability of HSP70 would be anticipated in TNFR-I KO splenocytes. Moreover, no additional bands of smaller molecular size were observed by immunoblotting in TNFR-I KO Mphi following exposure to stress.

Because TNF-alpha induces HSP70 in several cell types and has been implicated as a mediator of heat stress-induced HSP70 expression (21), we anticipated that the decreased expression of HSP70 in the TNFR-I KO Mphi would be related to heat-induced TNF-alpha . Thus we examined Mphi TNF-alpha production following heat stress. Surprisingly, we found that TNF-alpha was not synthesized following heat stress in any genotype examined (wild type, TNFR I KO, and TNFR II KO), although all genotypes synthesized TNF-alpha in response to LPS stimulation. Thus the effect of TNFR-I on heat stress-induced Mphi HSP70 is independent of TNF-alpha . This notion is further supported by our observation that heat stress induction of HSP70 in TNF-alpha KO Mphi is similar to that in wild-type Mphi and by the lack of suppression of heat-induced HSP70 production by TNF bp in wild-type Mphi .

The results of the present study suggest that the TNFR-I regulates HSP70 production by both TNF-alpha -dependent and -independent pathways. In addition, TNFR-I regulation of Mphi HSP70 production occurs at the posttranscriptional level. Furthermore, this effect is cell-type dependent. Macrophages isolated from TNFR-I KO mice show attenuated HSP70 induction following stress, while TNFR-I KO splenocytes show stress-induced HSP70 that is similar to wild type. This differential effect may be related to cellular trafficking pathways unique to the macrophage, such as its role as an antigen-presenting cell. Macrophages are central to the inflammatory response, releasing the proximal mediators of the proinflammatory cascade, such as TNF-alpha . Splenocytes are composed of a heterogeneous cell population, with a majority being T cells that can be activated by macrophages. The primary role of the macrophage in the immune response may explain the differential response seen between splenocytes and macrophages. Alternatively, the heterogenous nature of the splenocyte population may be important.

A number of studies demonstrate that the regulation of HSP70 induction is mediated at the level of transcription via DNA binding of HSF1 (1, 20). In our study, however, comparable steady-state levels of HSP70 mRNA were detected by RT-PCR in wild-type and TNFR-I KO Mphi despite differences in protein expression. Our results suggest an additional level of complexity in the regulation of HSP70 induction, which appears to be at the posttranscriptional level. Posttranscriptional regulation of protein expression may occur at the ribosomal binding of eukaryotic initiation factors (9). An additional mechanism of regulation of protein expression is the rate of mRNA degradation (10). It is possible that differential mRNA expression may be seen over time, though stable HSP70 mRNA has been observed by multiple investigators (14, 26). Because HSP70 mRNA transcript production is similar in TNFR-I KO and wild-type Mphi , posttranscriptional regulation by TNFR-I may involve translational machinery. Heat stress leads to the inhibition of normal cellular protein mRNA translation, with the preferential translation of HSP mRNA. This differential regulation of protein expression may occur at the ribosomal binding of eukaryotic initiation factors (9). Eukaryotic initiation factor 4F (eIF-4F) has been demonstrated to rescue protein synthesis in cell-free systems prepared from heat-shocked cells, while translation of heat shock mRNA is not affected by the loss of eIF-4F and, thus, is a possible contributing mechanism for the preferential translation of HSP mRNA. The regulation of eIF-4F is incompletely understood, but perhaps TNFR-I activation plays a role. The mechanism by which TNFR-I regulates Mphi HSP70 expression remains to be defined, though investigation of normal cellular mRNA translation in TNFR-I KO macrophages following heat shock may be helpful in elucidating the potential role of initiation factors.


    ACKNOWLEDGEMENTS

We are grateful to Lihua Ao for technical assistance in immunoblotting.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants GM-49222, GM-08315, AI-15614, and AI-2532359.

Address for reprint requests and other correspondence: J. K. Heimbach, Dept. of Surgery, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: julie.heimbach{at}uchsc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 19 September 2000; accepted in final form 1 February 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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