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Original Studies |
Hospices Civils de Lyon, Laboratoire de la Clinique Endocrinologique, Hôpital de l’Antiquaille, 69321 Lyon, France; and INSERM U-329, Hôpital Debrousse, 69005 Lyon, France
Address all correspondence and requests for reprints to: Prof. Michel Pugeat, Clinique Endocrinologique, Hôpital de l’Antiquaille, 1 rue de l’Antiquaille, 69321 Lyon Cedex, France.
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Abstract |
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In culture medium from HepG2 cells, the immunoconcentration of CBG and
the levels of CBG messenger ribonucleic acid (mRNA) were dose
dependently decreased in the presence of IL-6 concentrations ranging
from 0.1–10 ng/mL. The percent decrease in CBG immunoconcentration was
quantitatively similar to the percent decrease in CBG mRNA levels
(29 ± 6% and 39 ± 15%, respectively, of control values).
In contrast, and as expected, IL-6 dose dependently increased the mRNA
levels (164 ± 22% of control values) of
1-antitrypsin, a positive acute phase protein, but did
not affect the immunoconcentration of sex hormone-binding globulin,
another liver protein.
Dexamethasone alone did not significantly affect CBG secretion or mRNA levels, but did dose-dependently increase tyrosine aminotransferase mRNA levels, which increased to 252 ± 16% of the control values. However, in combination with IL-6, dexamethasone had a significant additive effect on IL-6 inhibition of CBG secretion and mRNAs in HepG2 cells.
IL-1ß dose-dependently stimulated CBG secretion (156 ± 10% of control values) with no significant effect on CBG mRNA levels. In addition, IL-1ß significantly decreased the inhibitory effect of IL-6 on CBG secretion, but had no effect on the inhibitory effect of IL-6 on CBG mRNA levels. These results suggest that IL-1ß acts on the posttranslation processing and/or secretion mechanisms of CBG in HepG2 cells.
Together, the present results strongly support the hypothesis that the decrease in plasma CBG concentrations is associated with the increase in IL-6 and glucocorticoid levels reported in patients with septic shock and burn injury.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin (1-AT) as well as other
members of the serine protease inhibitor (serpins) superfamily (4).
These proteins (1-AT and other serpins) are
positive acute phase proteins because their synthesis rate dramatically
increases during the acute phase response to bacterial infection,
surgical and other trauma, burn injury, tissue infarction, and
inflammatory states. Conversely, in the same circumstances, albumin,
transferrin, and -fetoprotein show a characteristic decrease in
liver synthesis and are, therefore, called negative acute phase
proteins (5).
The cytokines interleukin-6 (IL-6) and IL-1ß are synthesized and secreted by stimulated human monocytes. They are regarded as the main mediators of acute phase protein induction in the liver (6, 7). In addition, it appears that cofactors, such as glucocorticoids, may participate in acute phase protein regulation by acting in synergy with cytokines to stimulate positive acute phase protein production by the liver (8).
CBG is produced by human hepatoma-derived (HepG2) cells (9, 10), and it has been demonstrated that IL-6 decreases CBG synthesis from HepG2 cells (11). In the present study, the effects of acute phase cytokines (IL-1ß and IL-6) and dexamethasone on the production of CBG by HepG2 cells were further investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Medium, cell culture reagents, and human transferrin were
purchased from Life Technologies (Grand Island, NY). Culture flasks
were obtained from Falcon Plastics (Oxnard, CA). Dexamethasone, cell
culture grade, was obtained from Sigma Chemical Co. (St. Louis, MO).
Human recombinant IL-6 (SA, 1 x 107 IU/mg) and human
recombinant IL-1ß (SA, 1 x 107 IU/mg) were
purchased from R&D Systems (Abingdon, UK). CBG and
1-AT (used as a control for the IL-6 effect)
cDNA human probes were provided by G. L. Hammond (London Regional
Cancer Center, London, Canada). Tyrosine aminotransferase (TAT) cDNA
human probe, used as a control for the dexamethasone effect, was a gift
from G. E. Seralini (Laboratoire de Biochimie, CHU
Côte-de-Nacre, Caen, France). Na125I and
[-32P]deoxy-CTP were purchased from Amersham
International (Aylesbury, UK). Chemicals were obtained from Merck
(Darmstadt, Germany).
Cell culture
The HepG2 cell line (HB 8065, American Type Culture Collection, Rockville, MD) was grown as previously described (10). Approximately 5 x 106 cells were plated in T-25 flasks with 5 mL medium supplemented with 10% FCS, L-glutamine (30 g/L), penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin B (0.25 µg/mL). Near confluence, cells were washed twice with phosphate-buffered saline (PBS) and reincubated in RPMI 1640, supplemented with human transferrin (10 µg/mL), HEPES (0.4 mmol/L), L-glutamine (30 g/L), penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin B (0.25 µg/mL) without FCS for 24 h and then for an additional 48 h in the same medium with or without hormones. For each concentration of each hormone and for controls, three culture flasks were prepared.
Hormone preparations
IL-6 and IL-1ß were diluted in PBS with 0.1% BSA and were added to serum-free medium at concentrations of 0.1–10 ng/mL. Dexamethasone (stock solution in ethanol, 2.54 x 10-3 mmol/mL) was used at final concentrations of 10-8-10-5 mmol/mL.
In experiments on combined effects, cells were incubated with IL-6 (5 and 10 ng/mL) and dexamethasone (10-6 mmol/mL). The effects of IL-6 (5 and 10 ng/mL) in combination with IL-1ß (5 and 10 ng/mL) were also studied. Controls, using support medium only, were performed.
After 48 h of incubation, the medium from one flask was removed, and the cells were used for total ribonucleic acid (RNA) extraction. The medium of the other two flasks was harvested and centrifuged at 3000 rpm, and the cell-free supernatant was kept at -20 C pending assays. The cells were rinsed twice with PBS, collected using 0.25% trypsin-ethylenediamine tetraacetate solution, washed, and resuspended in RPMI 1640 with 10% FCS. Cells were centrifuged (800 rpm, 5 min) twice in PBS to get rid of medium culture components that would interfere with the fluorimetric DNA assay. The pellets were resuspended in 10 mL PBS and stored at -20 C for DNA assay.
The percentage of trypan blue-positive cells was less than 5% at the end of culture for each concentration of cytokine and glucocorticoid studied.
CBG assay
CBG was measured by RIA, as previously described (12). The sensitivity of the assay (38 pmol/L) was sufficient to measure the amount of CBG secreted in the medium.
SHBG assay
SHBG secretion was used as a control for protein secretion. SHBG secreted into the medium was measured with a double monoclonal immunoradiometric assay kit (125I-SHBG COATRIA, BioMerieux, Marcy l’Etoile, France), with the modifications previously described (10).
DNA assay
DNA measurement was performed using an ethidium bromide fluorimetric technique and calf thymus DNA as a standard (13). As a preliminary, frozen-thawed cells were lysed by short ultrasonic disintegration in 10 mL PBS solution. Two hundred-microliter aliquots were used for assay. CBG and SHBG were expressed relative to cellular DNA content because the normalization of data in terms of cell number was difficult due to cell clustering.
Northern blots
Total RNA was extracted as previously described (10) according
to the published modification (14) of the method of Chomzynski and
Sacchi (15). Forty micrograms of total RNA were loaded on 1%
formaldehyde-agarose gel and transferred to a nylon membrane (Hybond-N,
Amersham) using a vacuum transfer system (Pharmacia, Uppsala, Sweden).
Ethidium bromide staining was used to check RNA preparation integrity
by demonstrating the presence of intact 18S and 28S ribosomal RNA
bands. RNAs were fixed on the membrane by baking for 2 h at 80 C.
Prehybridization, with 50% formamide, 0.75 mol/L NaCl, 20 mmol/L
NaPO4, 1 mmol/L ethylenediamine tetraacetate, 5 x
Denhart’s solution, 10% dextran sulfate, and 100 µg/mL salmon sperm
DNA, was performed for 2 h at 42 C. The human CBG probe was a cDNA
of 1.2 kilobases (kb) corresponding to the major part of the coding
region of human CBG (4). The 1-AT cDNA probe
was a 1.3-kb fragment containing the entire coding region of human
1-AT. The TAT probe was a 2-kb fragment
corresponding to the entire coding region of human TAT cDNA (16).
Hybridization was performed overnight at 42 C with
[32P]CBG,
[32P]1-AT, or
[32P]TAT cDNA probes (25 ng) labeled with
[-32P]deoxy-CTP using the random priming technique
(multiprime labeling system, Amersham). Membranes were washed for 15
min in 2 x SSC (standard saline citrate)-0.1% SDS at room
temperature, followed by 15 min in 0.5 x SSC-0.1% SDS at 65 C
and 10 min (for CBG cDNA probe), 20 min (for
1-AT cDNA probe), or 5 min (for TAT cDNA
probe) in 0.1 x SSC-0.1% SDS at 65 C. Membranes underwent
autoradiography for between 6–72 h at -70 C. Autoradiograms and 28S
RNA ethidium bromide fluorescence photographs were scanned using a
Onescanner (Apple system, Cuppertino, CA) with Otofoto and NIH Image
softwares, and the individual messenger RNA (mRNA) densitometer scans
were normalized onto the 28S densitometer scans.
Statistical analysis
Data are expressed as the mean ± SEM for a minimum of three independent experiments and were analyzed by ANOVA followed by the Bonferroni-Dunn posttest. P < 0.05 was considered statistically significant. Dose-dependent effects of IL-6 and IL-1ß were studied by the least square regression method using the logarithm of the dose.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dexamethasone (10-8-10-5 mmol/mL) did
not affect CBG mRNA levels, whereas TAT mRNA levels were significantly
and dose dependently increased (r = 0.92; P <
0.0001), with a maximum stimulatory effect of 252 ± 6% of
control values at 10-5 mmol/mL (Fig. 1A
). CBG secretion and SHBG secretion by
HepG2 cells were not modified by dexameth-asone.
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IL-6 increased 1-AT mRNA levels dose
dependently (r = 0.56; P = 0.04), with a maximum
effect at 1 ng/mL (164 ± 22% of control values; Fig. 2B).
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). The maximum inhibitory effect of IL-6 on CBG
secretion was obtained at 5 ng/mL (71 ± 6% of control values;
P < 0.0001) and plateaued at higher doses (20 and 30
ng/mL). IL-6 also decreased CBG mRNA levels dose dependently (r =
0.73; P = 0.004; Fig. 2B), with a maximum inhibitory
effect at 10 ng/mL (61 ± 15% of control values;
P < 0.0001). This inhibitory effect of IL-6 on CBG
production was not different after 24-h incubation.
SHBG secretion (Fig. 2A) and cellular DNA content (data not shown) were
not significantly affected by IL-6 at the doses used in this study.
Effects of dexamethasone and IL-6
Dexamethasone (10-6 mmol/mL) significantly enhanced
the inhibitory effect of IL-6 on both CBG concentrations (31 ±
4% vs. 22 ± 4% of control values; P
= 0.0004) and CBG mRNA levels (58 ± 5% vs. 48 ±
4% of control values; P = 0.04) at 10 ng/mL IL-6
(Table 1).
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1-AT mRNA (167 ± 9% vs.
127 ± 12% of control values; P = 0.04). Effect of IL-1ß
CBG and 1-AT mRNA levels were not
significantly affected by IL-1ß. IL-1ß (0.1–10 ng/mL) dose
dependently increased CBG secretion (r = 0.57; P
< 0.001; Fig. 3). The maximum
stimulatory effect of IL-1ß on CBG secretion was 157 ± 10% of
the control value at 5 ng/mL. SHBG secretion was not significantly
modified by IL-1ß at the doses used in this study (Fig. 3).
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CBG secretion and mRNA were decreased significantly at
concentrations of 5 and 10 ng/mL IL-6, whereas at these concentrations,
IL-1ß increased CBG secretion, but did not change CBG mRNA (Table 2). The combined effects of IL-6 and
IL-1ß at 5 ng/mL each resulted in unchanged CBG secretion but
decreased CBG mRNA; at 10 ng/mL each, the effects were increased CBG
secretion but decreased CBG mRNA (Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-AT mRNA levels, and
glucocorticoids increase TAT mRNA levels, in agreement with their known
regulation (6, 17). We report in this study that IL-6 dose dependently
inhibits CBG secretion and CBG mRNAs, and that dexamethasone has no
effect on CBG production by HepG2 cells. However, dexamethasone in
combination with IL-6 has a significant additive effect on the
inhibitory effect of IL-6 on CBG production. The regulation of CBG by glucocorticoids varies in mammal species, following a temporal pattern (3). For example, in sheep liver, CBG mRNA levels are increased by glucocorticoids during fetal life, then decreased during adult life (18); in the adult rat, liver CBG biosynthesis has been shown to be reduced by dexamethasone administration at the transcriptional level (19), whereas in the baboon, betamethasone treatment between early and midgestation elicits a significant increase in fetal, but not maternal, CBG concentrations (20). In human hepatoma cells, no apparent repression of CBG transcription by dexamethasone could be demonstrated in our study. This might be explained by the fact that HepG2 cells appear to behave more like fetal hepatocytes than differentiated adult hepatocytes and may have different transcriptional regulatory proteins from those of nontransformed hepatocytes.
The inhibitory effect of IL-6 on CBG production in HepG2 cells is consistent with the presence of a recognition sequence for the nuclear factor-IL-6-binding site in the proximal promoter of the rat CBG gene, which is conserved in the promoter of the human CBG gene (21). In HepG2 cells, IL-6 doses higher than 10 ng/mL failed to achieve greater CBG repression than did lower doses (11). This might be explained by the rapid down-regulation of IL-6-binding sites. Indeed, high doses of IL-6 lead to internalization of the IL-6-binding subunit of the IL-6 receptor on HepG2 cells (22). The IL-6 receptor has two differentially regulated components: the IL-6-binding subunit, or gp80, and the IL-6 signal transducing protein, or gp130. The expression of gp80 on HepG2 cells is relatively low, but increases after treatment with dexamethasone (23). Additionally, a combination of IL-6 plus dexamethasone increases gp130 mRNA levels approximately 5-fold, although dexamethasone alone has no effect (24). The additional inhibitory effect of IL-6 plus dexamethasone on CBG production by HepG2 cells is, therefore, most likely explained by increasing expression of IL-6 receptors.
IL-1ß stimulated CBG secretion, but did not change CBG mRNA levels in HepG2 cells. In addition, when IL-6 is used in combination with IL-1ß, the CBG mRNA level is decreased to the same level as that with IL-6 alone, and the CBG concentration in culture medium is still increased, but less than by IL-1ß alone. These results suggest that, conversely to IL-6, which decreases CBG mRNA levels, IL-1ß acts on the posttranslation processing and/or the secretion mechanism of CBG. These opposite effects of IL-1 and IL-6 on the expression of acute phase proteins by HepG2 cells have previously been reported (25). Although the regulation of acute phase proteins might differ between normal and malignant cells (26), our results illustrate that CBG is a negative acute phase protein differentially regulated by IL-6 and IL-1ß.
In previous clinical studies, we reported that the concentration of CBG is dramatically decreased in patients with septic shock (12, 27). These results were in agreement with the initial observation by Savu et al. (28) that protein-binding activity for cortisol was decreased in serum from septic shock patients. In severe burn patients, the dramatic increase in free urinary cortisol reported was in contrast with slight increases in the cortisol concentration (29). This apparent discordance was explained by a large decrease in the CBG concentration in burn patients (29). Interestingly, the increased bioavailability of cortisol was related to higher protein breakdown and could also explain impaired immune function and delayed wound healing, suggesting that glucocorticoids might have deleterious effects during burn injury as well as in sepsis and shock.
The prolonged decrease in CBG concentration that we reported in septic shock patients (12) or in burn patients (29) suggests that hepatic production of CBG might be inhibited. Our present data suggest that the inhibitory effect of IL-6 on CBG production could maintain the low plasma CBG concentration in patients with septic shock before recovery (12) or with burn injury before wound healing (29). Our preliminary results showing a highly significant correlation between increased IL-6 and decreased CBG levels in severely burned patients (30), submitted) suggest that IL-6 might be an important regulator of the CBG level in humans during severe stress and a target for further treatment of these patients.
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Acknowledgments |
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Footnotes |
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Received May 7, 1997.
Revised July 2, 1997.
Accepted August 6, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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