AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 290: G903-G911, 2006. First published December 8, 2005; doi:10.1152/ajpgi.00089.2005
0193-1857/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/G903    most recent
00089.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wigmore, S. J.
Right arrow Articles by Garden, O. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wigmore, S. J.
Right arrow Articles by Garden, O. J.

INFLAMMATION/IMMUNITY/MEDIATORS

Febrile-range temperature but not heat shock augments the acute phase response to interleukin-6 in human hepatoma cells

Stephen J. Wigmore,1,2 Kenneth C. H. Fearon,1 James A. Ross,1 Stephen J. McNally,1 William J. Welch,2 and O. James Garden1

1Tissue Injury and Repair Group, MRC Centre for Inflammation Research, Medical School, University of Edinburgh, Teviot Place, Edinburgh, United Kingdom; and 2Surgical Research Laboratory, University of California San Francisco, San Francisco General Hospital, San Francisco, California

Submitted 24 February 2005 ; accepted in final form 5 December 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The relationship between the stress protein response and the acute phase response (APPR) was studied in human hepatoma cells to investigate the hierarchy of regulation of these survival responses. Huh-7 cells were subjected to heat treatment (febrile-range temperature 40°C or heat shock 43°C) followed by recovery at 37°C in the presence or absence of IL-6 given either before or after heat treatment. The effects on total, fractional, and acute phase protein synthesis were then analyzed by metabolic labeling, ELISA, real-time PCR, Northern blot analysis, and activation of an {alpha}1-antitrypsin reporter plasmid. Cell energetics were studied under the same conditions using an index of mitochondrial activity and measurement of cellular ATP levels. Febrile-range temperature (40°C) augmented acute phase protein production when cells had been pretreated with IL-6. Pretreatment of cells with IL-6 also prevented heat shock-induced suppression of {alpha}1-antichymotrypsin (ACT) but not transferrin. mRNA expression of ACT and {alpha}1-antitrypsin reporter activation studies was consistent with transcriptional regulation of these proteins. Expression of mRNA transcripts for transferrin was increased despite protein expression being reduced by heat shock. The effects of heat shock on acute phase protein synthesis can be modified by preincubation with IL-6, whereas addition of this ligand after heat treatment has no effect on the suppressive effect of heat on the APPR. The mechanism of this action appears to be transcriptionally regulated in the case of ACT, but in the case of transferrin, it may be mediated by another process such as posttranslational modification.

{alpha}1-antitrypsin; hepatocyte; acute phase protein; stress response; {alpha}1-antichymotrypsin; transferrin

FEVER IS A COMMON MANIFESTATION of inflammation or infection in vertebrates. It is presumed that fever offers some survival advantage, however, the mechanism of the beneficial effect of fever is uncertain (8). Increasing the body temperature of mammals and fish has been demonstrated to confer a survival advantage against normally lethal viral and bacterial pathogens (2, 4, 13). The febrile response is associated with increased circulating concentrations of cytokines including TNF, IL-1, and IL-6 (18–19). Conversely, fever exerts complex effects on the expression of proinflammatory cytokines by different cell types (30).

Elevation of body temperature to various sublethal levels also initiates an intracellular defense mechanism mediated by production of the heat-shock and glucose-regulated proteins, known collectively as stress proteins. The stress protein response is highly conserved in teliological terms, and cells surviving the initial injury often develop increased resistance to subsequent injury, a phenomenon known as "thermotolerance" or "stress protein preconditioning" (33). Understanding and manipulating the thermotolerant phenotype has potential clinical benefits particularly in situations such as transplantation, elective surgery, and medicine in which an ischemia-reperfusion injury is anticipated (26).

The acquisition of thermotolerance may be associated with a temporary loss of specialized cell function, and this could be detrimental if, in the case of hepatocytes or renal tubular cells, this function is important for the well being of the organism (35). The hepatocyte is a professional secretory cell that exhibits many specialized functions important for survival of the organism. One such function is the production and secretion of acute phase proteins (APP) (5). This large family of proteins includes proteins important for opsonization of bacteria, binding lipopolysaccharide, maintenance of coagulation pathway antiproteases, maintenance of plasma oncotic pressure, and carriage of metal ions (7, 11, 39).

The cytokines involved in the mediation of fever and the systemic response to it mediate other cellular events, and one of these is the elaboration of APPs by the liver (17). A number of ligands is known to modulate expression of APPs (3, 5, 36); the most important of these is IL-6 (3, 20–21). The concentration of certain APPs changes in response to IL-6 treatment; those that increase are known as positive APPs, whereas those that decrease are termed negative APPs (5, 10).

The acute phase response is believed to be important in regulating systemic inflammation and can be considered as a systemic survival response. The nature and hierarchy of the relationship between the stress protein (cell survival response) and acute phase (systemic survival response) protein responses are not fully understood and are important for future preconditioning strategies directed at improving hepatocyte function. The purpose of this study was to investigate the effect of thermal stress on hepatocyte APP production in the presence and absence of the ligand IL-6 using human hepatoma cell lines as a model.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell Culture and Heat Shock

HUH-7 and HepG2 cells were obtained from the European Cell Culture Collection (Porton Down, UK) and were cultured in DMEM supplemented with 10% FBS, penicillin (50 IU/ml), and streptomycin (50 µg/ml) at 37°C in 95% humidified air and 5% CO2. Cells were used in the logarithmic phase of growth, and cells were subjected to heat shock at 40 or 43°C for 45 min when they were 60–80% confluent in shallow trays of water in a dedicated incubator in which CO2 had been calibrated and adjusted to account for increased temperature. After heat shock, the medium of cells was replaced and cells were allowed to recover for variable periods of time at 37°C. Recombinant human IL-6 10 ng/ml was added to cells either 2 h before or immediately after heat treatment. All experiments were performed using Huh-7 cells. Hep G2 cells were used to confirm the effects of IL-6 and febrile-range temperature or heat shock on APP production.

Measurement of Total, Secreted, and Cellular Protein Production

Cells were washed in methionine-free DMEM for 1 min and then labeled with 50 µCi [35S] methionine for 4 h. The label was then replaced with DMEM containing 2% FBS. Cells were then exposed to heat shock at 40 or 43°C for 45 min. Medium and cell lysate were collected over 24 h. Proteins were precipitated using trichloroacetic acid and solubilized. Aliquots were run on 12.5% SDS PAGE and also counted in scintillant.

Cell Survival and Gp130 Receptor Expression

Subconfluent (60–80%) cells were exposed to heat shock for 45 min at 40 or 43°C and then harvested immediately or allowed to recover for 12 or 24 h at 37°C. Medium and trypsinized cells were combined to include any cells which might have detached and were stained with propidium iodide. Cell populations were analyzed by flow cytometry and gated for propidium dim (apoptotic) and propidium bright (necrotic) populations. Verification of the status of these gated populations had been previously confirmed using dual Annexin V staining and BOB78, an antibody recognizing a cell-surface moiety only expressed during apoptosis (8). Huh-7 cells were labeled with Gp130 (IL-6 receptor) antibody and FITC-conjugated secondary antibody following heat treatment at 40 or 43°C and recovery at 37°C for 2 or 24 h.

Mitochondrial Activity

Hepatoma cells that had been exposed to heat shock at 40 or 43°C were incubated for 2 h in darkness in the presence of Alamar blue, which is oxidized by actively metabolizing cells to a pink color. Cell supernatants were harvested, and color change was measured at 540 nm and compared with 37°C controls.

Intracellular ATP Assay

Cell lysates were prepared from cells exposed to heat shock and normothermic controls and were kept on ice. To investigate the effect of inhibition of protein synthesis on ATP depletion, some cells were treated with 5 µg/ml actinomycin D or 7.5 µg/ml cycloheximide for 4 h before heat shock. Substantive reduction in protein synthesis in cells treated with actinomycin D or cycloheximide was confirmed by pulse-labeling cells with [35S]methionine (data not shown). Cellular ATP was measured using a dual-injection port luminometer by conversion of luciferin to oxyluciferin, pyrophosphate, and light.

APP Analysis

The APPs, ACT, and transferrin (TRF) were measured in cell supernatants diluted 1:10 by sandwich ELISA using specific paired antibodies recognizing different epitopes of the two APPs studied (DakoCytomation, Ely, UK). The secondary antibodies were horseradish peroxidase conjugated, the chromogen was orthophenylenediamine, and the reaction was stopped with 1 M H2SO4. Sample concentrations were calculated against standard curves using human serum protein calibrator (DakoCytomation). The interassay coefficient of variation was 10% for both assays, and intra-assay coefficient of variation was 6% for ACT and 9% for TRF.

Real Time PCR for APP mRNA

RNA isolation and fluorescence detection real-time PCR. RNA extraction and purification was performed according to the manufacturer’s instructions using TRIzol (Invitrogen, Paisley, UK). RNA samples were treated with DNAase and then run as a template for a standard PCR reaction using beta-actin primers to exclude the presence of contaminating DNA. RNA was reverse transcribed using avian myeloblastosis virus RT (Promega, Southampton, UK) and random decamers (Ambion, Huntingdon, UK). Fluorescence-detected real-time PCR was then performed using primers and probes specifically designed for human ACT (serpina 3): forward 5'-TTACTGAGGCAGAAATTCACTCA, reverse 5'-CAGCTCATCGCTGGAACTGAT, and TAMRA-labeled probe 6-FAM-TCCAGCACCTCCGCGCACC-TAMRA and TRF forward 5'-TGGTCCCAGTGTTGCTTGTG, reverse 5'-GCTTCGTTTGCCGCAATG, and probe 6-FAM-AGAAAGCCTCCTACCTTGATTGCATCAGGG-TAMRA. A standard reaction contained 12.5 µl Taqman universal master mix (ABPI Biosystems, Warrington, UK), 7 µl primer probe mix (25 µM primers, 5 µM probe, 1.25 µl 18s primer probe mix, and 1.75 µl water), and 2.5 µl cDNA template. Samples were run on an ABPI Taqman and analyzed using Sequence Detector 7.1 (Applied Biosystems).

Northern analysis of APP mRNA. Probes were constructed for ACT and TRF. Primers were designed to give products of 836 bp for ACT, forward 5'-AGGCAGAAATTCACTCAGAGCT and reverse 5'-CTAATGCAGAAAGGAGGGTGAT and 531 bp for TRF, forward 5'-GATAGTGGCTCCAGATGAACC and reverse 5'-CCCATGAGGAGAGCTGAATAGT. Total RNA isolated from SKHep cells was reverse transcribed using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, and PCR was then performed. Products were run on 0.8% agar gels containing ethidium bromide to confirm the presence of a single product, gel purified, and ligated into the pCR Topo 2.1 cloning vector (Invitrogen). Transformed competent E. coli (Invitrogen) were selected by disruption of the lacZ gene on plates containing 50 µg/ml ampicillin and coated with X-gal. Correct insertion of the probes was confirmed by restriction mapping using sacI for ACT (fragments 4465 and 239 bp) and apoI for TRF (fragments 3224, 658, and 531 bp). Maxipreps (Qiagen, Crawley, UK) were then prepared of the probes further confirmation of their authenticity was obtained by sequencing using the sequenase polymerase technique, which confirmed >98% homology with published sequences (data not shown). RNA isolated from hepatocytes treated with IL-6 and or heat shock at either 40 or 43°C was loaded onto urea gels and was visualized with ethidium bromide and assessed for equality of loading based on the 18s and 28s bands. Gels were transferred on to nylon membranes and crosslinked with ultraviolet light. DNA probes were labeled with [32P]dCTP using Klenow and hybridized for 16 h at 68°C.

{alpha}1-Antitrypsin Expression

Subconfluent (60%) Huh-7 cells were transiently transfected with a plasmid containing the {alpha}1-antitrypsin (AT) promoter upstream of beta-galactosidase (kind gift of Connie Myers, Univ. of California San Francisco) using 6 µl Fugene (Roche) to 1 µg DNA. Transfection efficiency was assessed by flow cytometry by cotransfection with pYFP yellow fluorescent protein expression vector and was an average of 22%. Empty vector and nontransfected controls were also used. Cells were exposed to 1 ng/ml IL-6 for 1 h before heat shock and were lysed after 24 h recovery at 37°C. beta-Galactosidase was measured using an enzymatic assay using o-nitrophenyl-B-D-galactopyranoside (Promega), and samples were read with a spectrophotometer at 420 nm.

Analysis

Results are presented as means and SD. Comparisons between groups of experiments were performed using ANOVA using SPSS v11.0 software (SPSS, Chicago, IL). A P level of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of Thermal Stress on Huh-7 Cell Protein Production

Huh-7 cells that were subjected to heat shock at 43°C demonstrated increased accumulation of [35S]methionine-labeled cellular protein production but reduced secreted protein production as determined by trichloroacetic acid precipitable methionine counts (P < 0.02; Fig. 1, A and C). This effect occurred both early after heat shock and in cells allowed to recover at 37°C for 24 h. In cells subjected to a 40°C heat treatment, however, the increase in intracellular protein production was less than that observed at 43°C, and secreted protein concentrations were increased compared with both control and 43°C heat-shocked cells (P < 0.05; Fig. 1, B and C). This effect was greatest early after 40°C heat treatment but was maintained in cells allowed to recover at 37°C for 24 h.


Figure 1
View larger version (43K):
[in this window]
[in a new window]
  		Fig. 1. Differential effect of febrile-range temperature and heat shock on intracellular and secreted proteins in hepatocytes. Cells labelled with [35S]methionine were exposed to heat treatment at 40°C or 43°C and harvested at 4 or at 24 h. Representative autoradiograms of intracellular proteins (A) and secreted proteins (B) are shown alongside trichloroacetic acid (TCA) precipitable counts expressed as a percentage of 37°C controls (C). *P < 0.05, {dagger}P < 0.01 compared with 37°C controls (ANOVA). Hsp, heat shock protein.

 
Effect of Thermal Stress and Heat Shock Protein 70 Expression

Addition of IL-6 to cells at 37°C had no effect on heat shock protein (HSP)70 expression (Fig. 2A). Febrile-range temperature resulted in a small but significant increase in HSP70 expression, but this was less than that observed following heat shock treatment of cells. Addition of IL-6 to cells before heat treatment increased heat-induced expression of HSP70 both in cells treated with febrile-range temperature (P < 0.05) and heat shock (P < 0.05; Fig. 2, B and C).


Figure 2
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2. IL-6 augments expression of HSP70 in response to heat in human hepatoma cells. Cells were treated with 10 ng/ml IL-6 at 37°C (A) and before exposure to heat (B) at the times indicated. HSP70 was measured in cell lysates by Western blot analysis and measured by densitometry, representative blot, and actin loading control (C) . *P < 0.05 vs. untreated same-temperature controls. IDV, integrated density volume.

 
Effect of Thermal Stress on Cell Survival, Cellular ATP, and Mitochondrial Activity

Heat shock treatment at either 40 or 43°C for 45 min had no demonstrable effect on cell survival at any time point studied (Fig. 3A). Mitochondrial activity measured by reduction of Alamar blue was significantly increased in heat-treated cells at 40 and 43°C compared with normothermic controls (P < 0.05) immediately following heat shock but returned to normal levels with 24 h of recovery at 37°C (Fig. 3B).


Figure 3
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3. Cell toxicity, mitochondrial activity and ATP in cells exposed to heat. A: cells exposed to heat for 45 min were allowed to recover for 12 h before trypsinization and staining with propidium iodide. Flow cytometry was performed. B: mitochondrial activity measured by Alamar blue conversion measured at 540 nm as described in MATERIALS AND METHODS. C: ATP concentrations in cell lysates measured by luminometry based on an ATP dependant-luciferase reaction as described in MATERIALS AND METHODS. ATP was measured 4 or 24 h after heat treatment. Some cell groups were pretreated with inhibitors of protein synthesis, actinomycin D or cycloheximide. *P < 0.05, {dagger}P < 0.01 vs. 37°C controls (ANOVA).

 
Depletion of ATP occurred following heat shock at 40 (P < 0.05) and 43°C (P < 0.01) compared with cells at 37°C (Fig. 3C). When protein synthesis was inhibited by incubation of hepatocytes with actinomycin D or cycloheximide, no significant change in ATP concentration was noted in cells incubated at 37°C. By contrast, the ATP depletion observed in hepatocytes exposed to heat shock at 40 or 43°C could be partially inhibited by prior incubation with actinomycin D or cycloheximide (Fig. 3C). Gp130 receptor expression was detected at a relatively low level in Hu-7 cells and was unchanged by heating cells to 40 or 43°C followed by recovery for either 2 or 24 h (data not shown).

Effect of Heat Shock on APP Production

Concentrations of ACT in the supernatants of cells exposed to febrile-range temperature at 40°C were transiently reduced and were followed by a period of recovery characterized by normalization and augmented secretion peaking 48 h after heat shock (Fig. 4A). The addition of IL-6 resulted in increased concentrations of ACT at 37°C (P < 0.05). Pretreatment of cells with IL-6 before exposure to 40°C resulted in significantly increased ACT concentrations compared with cells exposed to the same temperature in the absence of the ligand (P < 0.01) or with 37°C controls, which had been treated with IL-6 (P < 0.02; Fig. 4A). By contrast, ACT concentrations in supernatants from cells treated with 43°C heat shock were reduced immediately and remained significantly lower than 37°C controls for 48 h (Fig. 4B). Preincubation of IL-6 to cells resulted in increased secretion of the positive APP ACT compared with normothermic controls, and this was not suppressed following 43°C heat treatment. Addition of IL-6 to cells after heat treatment either at febrile-range temperature or heat shock had been initiated had no influence on the secretion of ACT (Fig. 4, A and B) compared with the effect of heat treatment alone.


Figure 4
View larger version (9K):
[in this window]
[in a new window]
  		Fig. 4. Effect of heat and IL-6 on {alpha}1-antichymotrypsin (ACT) production. A and B: cells were either exposed to 10 ng/ml IL-6 for 2 h before or 2 h after a 45-min heat shock stimulus. Media aliquots were collected for a 4-h period up to each of the time points indicated. ACT concentration in supernatants was measured by ELISA as described in MATERIALS AND METHODS. *P < 0.05, {dagger}P < 0.01, **P < 0.005, {dagger}{dagger}P < 0.001 vs. 37°C controls (ANOVA).

 
Concentrations of TRF were unchanged 4 h after treatment with febrile-range temperature and were increased by 24 h of recovery at 37°C compared with untreated cells (Fig. 5A). Addition of IL-6 to cells produced the predicted reduction in TRF expression compared with untreated controls. Febrile-range temperature augmented the suppressive response to IL-6 with peak suppression of TRF production 24 h after heat treatment (Fig. 5A). Treatment of cells with 43°C resulted in inhibition of TRF production compared with 37°C controls. This effect was again greater in cells that had been treated with IL-6 compared with untreated normothermic or heat-treated controls (Fig. 5B).


Figure 5
View larger version (9K):
[in this window]
[in a new window]
  		Fig. 5. Effect of heat and IL-6 on transferrin (TRF) production (A and B). Cells were either exposed to 10 ng/ml IL-6 for 2 h before or 2 h after a 45-min heat shock stimulus at either 40°C (A) or 43°C (B). Media aliquots were collected for a 4-h period up to each of the time points indicated. TRF concentration in supernatants was measured by ELISA as described in MATERIALS AND METHODS. *P < 0.05, {dagger}P < 0.01, **P < 0.005, {dagger}{dagger}P < 0.001 vs. 37°C controls (ANOVA).

 
Further analysis demonstrated that the patterns of the positive APP haptoglobin and the negative APP prealbumin followed very similar patterns to those of ACT and TRF respectively (data not shown). Similarly, experiments undertaken with the HepG2 cell line confirmed the same effects observed in the Huh-7 cell line with respect to heat and IL-6 on secretion of ACT and TRF.

APP mRNA Expression Following Heat Shock

Expression of mRNA encoding the positive APP {alpha}1-antichymotrypsin was increased by IL-6 and also by febrile-range temperature at 40°C (Fig. 6A). The greatest stimulus for mRNA was when cells were treated with IL-6 and then exposed to febrile-range temperature. The same pattern of mRNA expression was observed using Northern blot analysis (Fig. 6C).


Figure 6
View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6. Effect of heat and IL-6 on acute phase protein mRNA expression (A and B). Cells were treated with IL-6 before exposure to 40 or 43°C heat for 45 min. RNA was isolated, and changes in ACT and transferrin mRNA expression were analyzed by real-time PCR as described in MATERIALS AND METHODS. C: Northern analysis was used to confirm the patterns of changes in mRNA expression observed by real-time PCR as described in MATERIALS AND METHODS. *P < 0.05, {dagger}P < 0.01, **P < 0.005, {dagger}{dagger}P < 0.001 vs. 37°C controls (ANOVA).

 
Exposure of cells to 40 or 43°C increased the expression of TRF mRNA (Fig. 6B). Preincubation with IL-6 reduced TRF mRNA expression compared with untreated 37°C controls, irrespective of the temperature to which cells were exposed. These patterns of expression were the same measured by real-time PCR and using Northern blot analysis (Fig. 6C).

Effect of IL-6 and Temperature on {alpha}1-AT Promoter Activation

Heating cells to febrile-range temperature (40°C) increased {alpha}1-AT promoter activity expression compared with 37°C controls (P < 0.02), whereas heat shock at 43°C reduced activity (P < 0.05; Fig. 7). Addition of IL-6 increased activation of the {alpha}1-AT promoter compared with transfected 37°C controls. Preincubation of cells with IL-6 before 40°C heat treatment resulted in significantly increased promoter activity compared with cells at 37°C that had been preincubated with IL-6 (P < 0.05). Preincubation of cells with IL-6 prevented the reduction in promoter activity induced by heat shock (43°C; P < 0.05).


Figure 7
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 7. Effect of heat and IL-6 on transcription of the {alpha}1-antitrypsin promoter. Huh-7 cells were cotransfected with a reporter plasmid containing the {alpha}1-antitrypsin promoter coupled with beta-galactosidase (beta-gal) and pYFP as a control expression vector. Cells were pretreated with 10 ng/ml IL-6 2 h before exposure to heat at 40 or 43°C for 45 min. Lysates harvested 4 h later were analyzed for beta-gal activity by spectrophotometry. *P < 0.05 (ANOVA).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The APP response incorporates many elements that are important for the pursuance and regulation of inflammation. In this respect, it can be considered a systemic survival response. The stress protein response is also a survival response, but one that operates at the level of the individual cell. The hierarchy of regulation and the relationship between stress and APPs is therefore of interest. Early reports suggested that expression of genes encoding HSPs excluded expression of genes encoding APPs (28). The same research group subsequently reported that expression of HSP72 was permissive of expression of the APP {alpha}1-AT in the pig (25). Subsequent studies (6) demonstrated that in an ischemia-reperfusion model, expression of HSPs resulted in attenuated but not eliminated expression of APPs. A further study in the mouse (24) found that febrile-range whole body hyperthermia alone had no direct suppressive effect on APP expression. The combination of administration of intraperitoneal lipopolysaccharide followed by whole body hyperthermia, however, resulted in augmented production of IL-6 and APPs in the BALB/c mouse but not in C57 B6 mice, indicating that in the mouse, at least there may be strain-specific effects (24). A further study using the HepG2 human hepatocyte in a similar model to that described in the present study found that febrile-range temperature significantly upregulated expression of the positive APP haptoglobin in the absence of IL-6. Furthermore, the addition of IL-6 further augmented expression of haptoglobin compared with cells treated with febrile-range temperature or IL-6 alone (14).

The present study has investigated the relationship between these two cellular pathways in relationship to intensity of heat shock stimulus, the APP-stimulating cytokine IL-6, and time in the Huh-7 hepatoma cell line. Key experiments were confirmed using the HepG2 human hepatoma cell line. In the absence of IL-6, it was found that inhibition of APPs was proportional to the magnitude of the heat shock stimulus, i.e., inhibition was greater at 43°C than at 40°C (Figs. 4 and 5). This effect occurred at the same time that HSP expression was maximal, it and concurs with previously published reports of APP expression early after heat shock (6, 28). Labeling studies demonstrated that a large proportion of total cellular protein synthesis was diverted to the synthesis of HSPs (Fig. 1), and it is possible that in the early phase, heat shock results in a reprioritization of cellular protein synthesis toward cytoprotective stress proteins. Such cellular activity may also explain the observed increase in mitochondrial activity and ATP depletion observed following heat treatment.

Interestingly, cells exposed to febrile-range temperature (40°C) exhibited augmented synthesis of secreted proteins including APPs (Fig. 1). The profile of expression of APPs in relationship to time demonstrated early attenuation of APP secretion after heat shock followed by a period of recovery and augmented expression. The augmentation of APP expression following heat treatment was greatest after exposure to febrile-range temperature (Figs. 4 and 5). The time delay between suppression and normalization or augmented production of APPs was also proportional to the magnitude of the thermal stress. The majority of studies that have addressed the expression of APPs after heat shock have concentrated on the first 24 h after heat shock and have therefore not described this phenomenon. All of the experiments performed in this study were undertaken in the presence of heat-inactivated serum because of the increased sensitivity of cells to heat in the absence of serum; therefore, an effect of factors in the serum itself cannot be excluded.

The conventional classification of APPs into positive and negative reactants dependent on their response to IL-6 (3) has been brought into question. We have previously described various effects on the response of APPs to IL-6 depending on the combination of counterregulatory hormones present in the cell environment (23). We have also demonstrated that the presence of n-3 and n-6 fatty acids in culture medium can similarly alter the profile of expression of APPs in isolated human hepatocytes (37). Furthermore, the cytokine milieu arising from inflammatory cells in different disease states can influence the acute phase response (38). In the present study, responses of ACT and TRF to IL-6 treatment were as expected compared with normothermic nontreated controls. However, the response of cells to heat treatment in the presence of IL-6 was quite different with augmented expression of TRF compared with normothermic controls, which were not normalized by IL-6.

The acute phase response can be mediated by a number of cytokines (3, 5, 36); however, it is generally agreed that IL-6 is the principal cytokine involved in hepatic APP gene expression (10), and its signaling pathway has been well characterized (15, 20–21). Administration of IL-6 following heat shock altered the profile of both ACT and TRF expression in response to heat shock, and this suggests that the inhibitory effects of heat shock on APP synthesis are not absolute and can be modified. Furthermore, we have shown that the effect of IL-6 is entirely dependent on whether it precedes or follows heat treatment. When given before heat treatment, its effects in upregulating ACT are enhanced by heat treatment; however, if given afterward, it is the effect of heat treatment that predominates. This suggests that in this model, the effects of the acute phase response are subordinate to those of heat but can be modified to some extent by preincubation of cells with IL-6. In a biological systems correlate, it may be that an acute phase response initiated by IL-6 before the onset of fever might be enhanced by the fever.

The mechanism of APP suppression in relationship to thermal stress is unclear. Changes in expression of ACT mRNA in heat-shocked cells are compatible with transcriptional regulation (Fig. 6A); however, this may not be the case for TRF (Fig. 6B). Expression of mRNA transcripts for TRF increased in heat-shocked cells both by real-time PCR and Northern blot analysis at the time when there was reduced production of this protein; this may suggest the involvement of a posttranscriptional regulatory pathway (Fig. 6, B and C).

Expression of many APPs is known to be stimulated by IL-6 acting through nuclear factor (NF) IL-6. Interaction between NFIL-6 and heat shock transcription factor-1 has been previously reported in relationship to the heat shock 90beta promoter (31). In that circumstance, IL-6 augments the effect of HSF-1 on activation of the hsp90beta promoter, and this effect can be inhibited by STAT-3. This provides a mechanism by which the hsp90beta promoter could be regulated differentially in stressed and unstressed cells (31). The mouse CRP promoter contains cis-acting elements very similar to the heat shock element as well as several binding sites for the IL-6 nuclear factor (16, 22). It is possible that certain acute phase promoters may be subject to similar regulation as the hsp90beta promoter. Recent evidence suggests that a number of factors may come into play in the regulation of the hepatic APP response (32, 34) and that heat initiates a number of cellular events that can inhibit (40) or maintain APP expression (29) in different circumstances.

It is conceivable that some of the changes observed in this study might be attributable to the effect of heat on IL-6 receptor expression. We did not observe any changes in surface expression of the gp130 receptor but did not investigate the 80-kda IL-6 {alpha}-chain receptor. It has been demonstrated in the Hep G2 cell line that certain physiological events can alter expression of the 80-kda IL-6 receptor (IL-6R) and that this, in turn, can modify the response of cells to IL-6 (12, 27). Although it seems unlikely that changes in receptor expression would influence APP expression in the absence of ligand and in vivo data suggests no link between soluble IL-6R and APP expression in models of chronic inflammation (1), we have not excluded the possibility that changes in 80-kda IL-6R could contribute to the ligand-associated changes in APP production.

The exclusion of a biological pathway important for regulation of systemic inflammation to mount a cell-survival response would seem to be undesirable and biologically disadvantageous. This study confirms previous investigations demonstrating early suppression of the APP response in cells mounting a stress-protein response but shows that this suppression resolves and is followed by a variable period of augmented APP production. Furthermore, we have shown that the APP response to heat treatment can be modified by incubation of cells with the ligand IL-6 provided that this precedes the heat stimulus. The changes in expression of the positive acute phase reactant ACT appear to be transcriptionally regulated; however, there is a mismatch in the case of the negative acute phase reactant TRF such that heat induces increased mRNA expression but reduced protein expression. This study provides a model in which the APP response and stress protein responses may coexist at biologically relevant temperatures in a way that may be advantageous to the organism in terms of self-protection and innate immunity to infection in the presence of fever.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
S. J. Wigmore was supported by the Wellcome Trust Grant 065029. This project also received funding from the Union Internationale Contre Le Cancer Yamagiwa Yoshida Fellowship, Pathological Society of Great Britain and Ireland and Scottish Hospital Endowments Research Trust Travelling Fellowships.


   ACKNOWLEDGMENTS
 
The authors thank K. Sangster, A. Kabling, and Dr W. J. Hansen for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Wigmore, Liver Research Group, Univ. of Birmingham, Institute of Biomedical Research 5thFloor, Wolfson Drive, Birmingham B15 2TT, UK (e-mail: s.wigmore{at}bham.ac.uk)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Barber MD, Fearon KC, and Ross JA. Relationship of serum levels of interleukin-6, soluble interleukin-6 receptor and tumour necrosis factor receptors to the acute-phase protein response in advanced pancreatic cancer. Clin Sci (Lond) 96: 83–87, 1999.[Medline]
  2. Bell JF and Moore GF. Effects of high ambient temperature on various stages of the rabies virus infection in mice. Infect Immun 10: 510–515, 1974.[Abstract/Free Full Text]
  3. Castell JV, Gomez-Lechon MJ, David M, Fabra R, Trullenque R, and Heinrich PC. Acute phase response of human hepatocytes: regulation of acute phase protein synthesis by interleukin-6. Hepatology 12: 1179–1186, 1990.[ISI][Medline]
  4. Covert JB and Reynolds WW. Survival value of fever in fish. Nature 267: 43–45, 1977.[CrossRef][Medline]
  5. Gabay C and Kushner I. Acute phase proteins and other responses to systemic inflammation. N Engl J Med 340: 448–454, 1999.[Free Full Text]
  6. Gingalewski C, Theodorakis NG, Yang J, Beck SC, and De Maio A. Distinct expression of heat shock and acute phase genes during regional hepatic ischemia-reperfusion. Am J Physiol Regul Integr Comp Physiol 271: R634–R640, 1996.[Abstract/Free Full Text]
  7. Gourine AV, Gourine VN, Tesfaigzi Y, Caluwaerts N, Van Leuven F, and Kluger MJ. Role of alpha(2)-macroglobulin in fever and cytokine responses induced by lipopolysaccharide in mice. Am J Physiol Regul Integr Comp Physiol 283: R218–R226, 2002.[Abstract/Free Full Text]
  8. Hasday JD and Singh IS. Fever and the heat shock response: distinct, partially overlapping processes. Cell Stress Chaperones 5: 471–480, 2000.[CrossRef][ISI][Medline]
  9. Hart SP, Jackson C, Kremmel LM, McNeill MS, Jersmann H, Alexander KM, Ross JA, and Dransfield I. Specific binding of an antigen-antibody complex to apoptotic human neutrophils. Am J Pathol 162: 1011–1018, 2003.[Abstract/Free Full Text]
  10. Heinrich PC, Castell JV, and Andus T. Interleukin-6 and the acute phase response. Biochem J 265: 621–636, 1990.[ISI][Medline]
  11. Hochepied T, Van MOlle W, Berger FG, Baumann H, and Libert C. Involvement of the acute phase protein alpha 1 acid glycoprotein in non-specific resistance to a lethal gram-negative infection. J Biol Chem 275: 14903–14909, 2000.[Abstract/Free Full Text]
  12. Holub MC, Hegyesi H, Igaz P, Polgar A, Toth S, and Falus A. Soluble interleukin-6 receptor enhanced by oncostatin M induces major changes in gene expression profile of human hepatoma cells. Immunol Lett 82: 79–84, 2002.[CrossRef][ISI][Medline]
  13. Jiang Q, Cross AS, Singh IS, Chem TT, Viscardi RM, and Hasday JD. Febrile core temperature is essential for optimal host defense in bacterial peritonitis. Infect Immun 68: 1265–1270, 2000.[Abstract/Free Full Text]
  14. Karlsson JOM, Yarmush ML, and Toner M. Interaction between heat shock and interleukin-6 stimulation in the acute phase response of human hepatoma (HepG2) cells. Hepatology 28: 994–1004, 1998.[CrossRef][ISI][Medline]
  15. Kishimoto Kira S, Narazaki M, and Taga T. Interleukin-6 family of cytokines and gp130. Blood 86: 1243–1254, 1990.
  16. Ku NO, Mortensen RF. Cloning and tissue-specific expression of the gene for mouse C-reactive protein. Biochem J 295: 379–386, 1993.[Medline]
  17. Kushner I. Regulation of the acute phase response by cytokines. Perspect Biol Med 36: 611–622, 1993.[ISI][Medline]
  18. Le May DR, Le May LG, Kluger MJ, and D’Alecy LG. Plasma profiles of IL-6 and TNF with fever-inducing doses of lipopolysaccharide in dogs. Am J Physiol Regul Integr Comp Physiol 259: R126–R132, 1990.[Abstract/Free Full Text]
  19. Le May LG, Otterness IG, Vander AJ, and Kluger MJ. In vivo evidence that the rise in plasma IL-6 folloing injection of a fever-inducing dose of LPS is mediated by IL-1 beta. Cytokine 2: 199–204, 1990.[CrossRef][Medline]
  20. Majello B, Arcone R, Toniatti C, and Giliberto G. Constitutive and IL-6-induced nuclear factors that interact with the human C-reactive protein promoter. EMBO J 9: 457–465, 1990.[ISI][Medline]
  21. Morrone G, Giliberto G, Oliviero S, Arcone R, Dente L, Content J, and Cortese R. Recombinant interleukin-6 regulated transcriptional activity of a set of human acute phase genes. J Biol Chem 263: 12555–12558, 1988.
  22. Ohnishi S, Maeda S, Nishiguchi S, Arao T, and Shimada K. Structure of the mouse C-reactive protein gene. Biochem Biophys Res Commun 156: 814–822, 1988.[CrossRef][ISI][Medline]
  23. O’Riordain MG, Ross JA, Fearon KCH, Maingay JP, Farouk M, Garden OJ, and Carter DC. Insulin and counterregulatory hormones influence acute phase protein production in the human hepatocyte. Am J Physiol Endocrinol Metab 269: E323–E330, 1995.[Abstract/Free Full Text]
  24. Ostberg JR, Taylor SL, Baumann H, and Repasky EA. Regulatory effects of fever-range hyperthermia on the LPS-induced acute inflammatory response. J Leukoc Biol 68: 815–820, 2000.[Abstract/Free Full Text]
  25. Pannen BHJ, Maeda K, Ayuse T, Brienza N, Revelly JP, Robotham JL, and Buchman TG. Hepatic heat shock and acute phase gene expression are induced simultaneously after celiotomy in the anesthetized pig. Anesthesiology 83: 850–859, 1995.[CrossRef][ISI][Medline]
  26. Patel A, van de Poll MCG, Greve JW, Buurman WA, Fearon KCH, McNally SJ, Ross JA, Garden OJ, Dejong CHC, and Wigmore SJ. Early stress protein gene expression during intermittent portal ischemia-reperfusion in human liver surgery. Transplantation 78: 1479–1487, 2004.[CrossRef][ISI][Medline]
  27. Paysant J, Blanque R, Vasse M, Soria C, Soria J, and Gardner CR. Factors influencing the effect of the soluble IL-6 receptor on IL-6 responses in HepG2 hepatocytes. Cytokine 12: 774–9, 2000.[CrossRef][ISI][Medline]
  28. Schoeniger LO, Reilly PM, Bulkley GB, and Buchman TG. Heat shock gene expression excludes hepatic acute phase gene expression after resuscitation from hemorrhagic shock. Surgery 112: 355–363, 1992.[ISI][Medline]
  29. Shah M, Patel Fried VA K, and Sehgal PB. Interaction of STAT-3 with caveolin-1 and heat shock protein 90 in plasma membrane raft and cytosolic complexes. Preservation of cytokine signalling during fever. J Biol Chem 277: 45662–45669, 2002.[Abstract/Free Full Text]
  30. Singh IS, Viscardi RM, Kalvakolanu I, Calderwood S, and Hasday JD. Inhibition of tumor necrosis factor-alpha transcription in macrophages exposed to febrile range tremperature. A possible role for heat shock factor-1 as a negative transcriptional regulator. J Biol Chem 275: 9841–9848, 2000.[Abstract/Free Full Text]
  31. Stephanou A, Isenberg DA, Akira S, Kishimoto T, and Latchman DS. The nuclear factor interleukin-6 (NFIL-6) and signal transducer and activator of transcription-3 (STAT-3) signaling pathways co-operate to mediate activation of the HSP90b gene by interleukin-6 but have opposite effects on its inducibility by heat shock. Biochem J 330: 189–195, 1998.[Medline]
  32. Wang Y, Ripperger J, Fey GH, Samols D, Kordula T, Wetzler M, Van Retten RA, and Bauman H. Modulation of hepatic gene expression by epidermal growth factor and src protein tyrosine kinases in murine and human hepatic cells. Hepatology 30: 682–697, 1999.[CrossRef][ISI][Medline]
  33. Welch WJ and Mizzen LA. Characterisation of the thermotolerant cell. II. Effects on the distribution of heat shock protein 70, intermediate filaments and small ribonucleoprotein complexes. J Cell Biol 106: 1117–1130, 1988.[Abstract/Free Full Text]
  34. Welm AL, McKey SL, Timchenko LT, Darlington GJ, and Timchenko NA. Translational induction of liver-enriched transcriptional inhibitory protein during acute phase response leads to repression of CCAAT/enhancer binding protein mRNA. J Biol Chem 275: 27406–27413, 2000.[Abstract/Free Full Text]
  35. Wigmore SJ, Fearon KCH, and Welch WJ. The metabolic cost of thermal stress on human hepatocytes: implications for differentiated cell function (Abstract). Proc Nutr Soc 60: 67, 2001.
  36. Wigmore SJ, Fearon KC, Maingay JP, Lai PB, and Ross JA. Interleukin-8 can mediate acute-phase protein production by isolated human hepatocytes. Am J Physiol Endocrinol Metab 273: E720–E726, 1997.[Abstract/Free Full Text]
  37. Wigmore SJ, Fearon KCH, Maingay JP, and Ross JA. Modulation of human hepatocyte acute phase protein production in vitro by n-3 and n-6 polyunsaturated fatty acids. Ann Surg 225: 103–111, 1997 .[CrossRef][ISI][Medline]
  38. Wigmore SJ, Fearon KCH, Maingay JP, Carter DC, and Ross JA. Il-4 mediated down regulation of the acute phase protein response in multiple organ failure and gastrointestinal cancer. Clin Science 95; 347–354, 1998.[Medline]
  39. Xia D and Samols D. Transgenic mice expressing rabbit C-reactive protein are resistant to endotoxaemia. Proc Natl Acad Sci USA 94: 2575–2580, 1997.[Abstract/Free Full Text]
  40. Xie Y, Zhong R, Chen C, and Calderwood SK. Heat shock factor-1 contains two functional domains that mediate transcriptional repression of the c-fos and c-fms genes. J Biol Chem 278: 4687–4698, 2003.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/G903    most recent
00089.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wigmore, S. J.
Right arrow Articles by Garden, O. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wigmore, S. J.
Right arrow Articles by Garden, O. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.