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Endocrinology Vol. 147, No. 5 2518-2525
Copyright © 2006 by The Endocrine Society

Elevated Glucose and Diabetes Promote Interleukin-12 Cytokine Gene Expression in Mouse Macrophages

Yeshao Wen, Jiali Gu, Shu-Lian Li, Marpadga A. Reddy, Rama Natarajan and Jerry L. Nadler

Diabetes and Hormone Center (Y.W., J.G., J.L.N.), University of Virginia, Charlottesville, Virginia 22908; and Department of Diabetes Endocrinology and Metabolism (S.-.L.L., M.A.R., R.N.), City of Hope National Medical Center, Duarte, California 91010

Address all correspondence and requests for reprints to: Jerry L. Nadler, Diabetes and Hormone Center, University of Virginia, Charlottesville, Virginia 22908. E-mail: jln2n{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inflammation is emerging as an important mechanism for micro- and macrovascular complication of diabetes. The macrophage plays a key role in the chronic inflammatory response in part by generating particular cytokines. IL-1ß, IL-6, IL12, IL-18, TNF{alpha}, and interferon-{gamma} are produced primarily in macrophages and have been associated with accelerated atherosclerosis and altered vascular wall function. In this study, we evaluated the effect and mechanism of high glucose (HG) on gene expression of these cytokines in mouse peritoneal macrophages (MPM). HG led to a 2-fold increase in the mRNA expression of these cytokines, with IL-12 showing the highest activation (5.4-fold) in a time-dependent (3–12 h) and dose-dependent (10, 17.5, and 25 mmol/liter) manner. The effects were specific to HG because mannitol and 3-O-methyl-glucose had no effect on cytokine mRNA expression. HG also increased IL-12 protein accumulation from MPM. We also explored the role of induced and spontaneous diabetes on inflammatory cytokine expression in MPM. Increases in expression in MPM of multiple inflammatory cytokines, including a 20-fold increase in IL-12 mRNA, were observed in streptozotocin-induced type 1 diabetic mice as well as type 2 diabetic db/db mice, suggesting that cytokine gene expression is increased by hyperglycemia in vivo. We next explored potential mechanisms of HG-induced increases in IL-12 mRNA. HG increased the activity of protein kinase C, p38 MAPK (p38), c-Jun terminal kinase, and inhibitory-{kappa}B kinase in MPM. Furthermore, inhibitors of these signaling pathways significantly reduced HG-induced IL-12 mRNA expression in MPM. These results provide evidence for a potentially important mechanism linking elevated glucose and diabetes to inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MACROPHAGE plays a central role in chronic inflammation in diabetes and atherosclerosis. Macrophages are a major source of proinflammatory cytokines including TNF{alpha}, (1), IL-1ß (2), IL-6 (3), IL-12, and IL-18. TNF{alpha}, IL-1ß (1, 2), and IL-6 are important cytokines involved in chemotaxis cell adhesion. Evidence shows that IL-12 is an important cytokine that can program T cells to develop into Th1 cells (4, 5). IL-12 and Th1 cell infiltration has emerged as an important pathway for autoimmune diabetes as well as chronic inflammation associated with atherosclerosis (5, 6). Reduction of IL-12 is associated with reduced atherosclerosis in the mouse (7, 8).

Macrophages of alloxan diabetic mice with moderate diabetes (300 mg/dl) produced significantly more IL-6 and TNF{alpha} (9). Recent data have also shown that high glucose (HG) treatment activates monocytes and induces an increase in gene expression of TNF{alpha}, IL-1ß, and monocyte chemoattractant protein (MCP)-1 in human THP-1 monocyte like cells (10). However, it is not clear whether HG affects cytokine expression in primary tissue macrophages. Furthermore, how HG affects cytokine gene expression in tissue macrophages has not been evaluated.

The present study was designed to examine the effect and mechanism of high glucose on mRNA expression of cytokines in mouse peritoneal macrophages (MPMs) from C57BL6 mice. We found that HG treatment time- and dose-dependently leads to a more than 2-fold increase in mRNA expression of multiple cytokines with a marked increase in IL-12 gene expression. These effects are specific to HG because mannitol and 3-O-methyl-glucose had no effect. We also studied the in vivo relevance of these findings. The mRNA expression of these cytokines is also markedly increased in MPMs from streptozotocin (STZ)-induced diabetic C57BL6 mice as well as type 2 diabetic db/db mice. This is the first study showing that HG can increase cytokine gene expression and protein production in primed macrophages. These results provide a new explanation for the inflammatory effects of HG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Male C57BL6/J (8–10 wk old) and male db/db (8–10 wk old) mice were obtained from Jackson Laboratory (Bar Harbor, ME). Thioglycollate, STZ, GF-109203X, and calphostin C were from Sigma (St. Louis, MO); SB-202190 from Biosource International (Camarillo CA); c-jun-N-terminal kinase (JNK) inhibitor 1 and JNK inhibitor 1 control peptide from Alexis Biochemicals (San Diego, CA); nuclear factor-{kappa}B (NF{kappa}B) inhibitor and NF{kappa}B inhibitor control peptide from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and RNeasy minikit from QIAGEN (Valencia, CA). Culture media and DMEM were from American Type Culture Collection (Manassas, VA), and RPMI 1640 and Hanks’ balanced salt solution were from Invitrogen (Carlsbad, CA) All animal studies were approved by our Institutional Animal Safety Committee.

MPM isolation and culture
Mice were injected ip with 2 ml 4% thioglycollate solution. Three days later, the ascites of these mice was collected and cells were spun down, resuspended in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin, seeded, and cultured on 100-mm plates for 3 h in 5% CO2 incubator at 37 C. Nonattached cells were carefully washed away by washing cells four times with Hanks’ balanced salt solution. The attached MPMs were resuspended in the same medium as above. MPM number was counted, seeded again on 60-mm culture dishes, and incubated overnight. The attached cells comprised more than 95% MPMs as reflected by specific macrophage antibody staining.

MPM treatment
The MPMs were serum depleted from typical RPMI 1640 medium containing 10 mmol/liter D-glucose (NG) supplemented with 10% heat-inactive FBS and penicillin/streptomycin for 24 h and were then cultured in varied glucose concentration for certain periods of time. In some experiments cells were pretreated with one of the following inhibitors: calphostin C, GF-109203X, SB-202190, JNK MAPK inhibitor, or NF{kappa}B inhibitor for 30 min. The cells were then incubated in basal or elevated glucose for 3 h in the presence of inhibitors.

Preparation of STZ-induced diabetic mice
A single STZ injection (freshly dissolved in saline) at dose of 280 mg/kg body weight was injected ip into C57BL6 mice. Blood glucose was measured with an Accu-Chek Advantage meter (Roche Diagnostics, Indianapolis, IN) and was expressed as milligrams per deciliter. To avoid the effect of STZ itself on cytokine expression, macrophages were isolated at least 10 or more days after STZ injection. All animal protocols were approved by the Institutional Animal Safety Committee of the University of Virginia.

RNA extraction and real-time PCR
Total RNA was extracted from cells using the RNeasy kit (QIAGEN), with deoxyribonuclease I treatment. The RNA was processed to cDNA synthesis using the SuperScript II reverse transcriptase and oligo d(T) (Invitrogen) according to the manufacturer’s protocol. The sequences of forward and reverse primers for target cytokine genes and house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were selected based on published sequence data from the National Center for Biotechnology Information database and previous study (11).

Primers were synthesized from Integrated DNA Technologies Inc (Coralville, IA). For quantitation, a double-stranded DNA dye, SYBR Green I (Molecular Probes Inc., Eugene, OR), was used along with AmpliTaq Gold and 0.1 µM of each primer. All reactions were performed in triplicate in iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). The real-time PCR was carried out in two steps: step 1, 94 C for 10 min, one cycle, and step 2, 94 C for 15 sec, followed by 60 C for 60 sec, 45 cycles. GAPDH was used as an endogenous reference to correct for differences in the amount of total RNA added to the reaction and compensate for different levels of inhibition during reverse transcription of RNA and during PCR. Data are calculated using the 2–{Delta}{Delta}CT method (12) and are presented as fold induction of transcripts for target genes normalized to GAPDH in cells treated with HG or inhibitors.

Total RNA of macrophages from db/db and control db/+ mice was extracted with Trizol Invitrogen according to manufacturer’s instructions. One microgram of total RNA was used for reverse transcription using the GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA). 18S primers were used as internal control. The software used for quantitation of PCR data were Quantity One (Bio-Rad).

Cytokine protein release measurement using ELISA
Cells were seeded on 24-well plates with each well containing 1 x 106 cells of MPMs in 1 ml medium. After serum depletion for 24 h, cells were then treated either with HG (25 mmol/liter D-glucose) or NG (10 mmol/liter D-glucose). Conditioned medium was collected in a sequential fashion (e.g. 0–16, 16–24, 24–36, and 36–60 h) with a change of 1 ml fresh medium containing HG or NG at each time point. The collected culture-conditioned medium was centrifuged to separate floating cells in the medium, and the supernatant was stored at –80 C. IL-12 protein levels were measured using a specific mouse IL-12 p40 immunoassay kit (R & D Systems, Minneapolis, MN).

Kinase activity measurement using Western blot with specific phosphoantibodies
Fifty micrograms HG-treated protein extracts were resolved on 10% SDS-polyacrylamide gel. Electrophoresis was run under constant voltage conditions. The proteins on the gel were transferred onto Immuno-Blot polyvinylidene difluoride membrane from Bio-Rad that was incubated consecutively with specific phosphoantibody and antirabbit or antimouse Ig conjugated with horseradish protein, and the protein bands were detected using enhanced chemiluminescence detection reagents from Amersham Biosciences Co. (Piscataway, NJ). The membrane was then stripped using Restore Western blot stripping buffer (Pierce Co., Rockford, IL), and the membranes were incubated with nonphosphoantibody or ß-actin and detected using enhanced chemiluminescence detection reagents. The result presented in the figures was a representative of a minimum of three experiments using three different batches of primary cultured macrophages treated with HG for different times.

Data analysis
The results are expressed as mean ± SEM from combined experiments as noted in each legend. For experiments running at one time period, the control and experimental samples are analyzed using the Student’s t test. These comparisons are based on a minimum of three experiments in triplicate per treatment. For multiple time periods of conditions, ANOVA with Duncan’s or Dunnett’s test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HG increases cytokine expression
Macrophages were incubated with RPMI 1640 medium containing10% inactive FBS for 24 h and then treated with NG or HG for the indicated amount of time. mRNA expression was measured with real-time RT-PCR in whole cell RNA extracts. Figure 1AGo shows that HG dose-dependently (10, 17.5, and 25 mmol/liter) increases the mRNA expression of multiple cytokines including IL-1ß, IL-6, IL-12, IL-18, and TNF{alpha} more than 2-fold over that in NG-treated macrophages. Of these cytokine genes, IL-12 mRNA expression was increased the most. D-glucose (17.5 mmol/liter) induced a 3.5-fold increase, whereas 25 mmol/liter D-glucose induced a 5.5-fold increase in IL-12 gene expression, compared with that under NG conditions (P < 0.01). HG also increases interferon-{gamma} about 2-fold (data not shown). Figure 1BGo shows the time course of HG-induced mRNA expression of these cytokine genes, which occurred as early as 3 h, and some reached a peak at 3 h and remained elevated for 6 h after HG treatment. The HG-induced increase in mRNA expression declined at 12 h of treatment. However, the value remained elevated, compared with that under the NG conditions (Fig. 1BGo). At 24 h of HG treatment, the HG-induced mRNA expression of most cytokine genes returned to the value seen under NG conditions (data not shown). To examine whether high osmotic pressure plays a role in HG-induced increase in cytokine gene expression, IL-12 was taken as an example. Macrophages were treated with 10 mmol/liter D-glucose + 15 mmol/liter of mannitol or 3-O-methyl-glucose (3OMG) for 3 h, and IL-12 gene expression was measured. Figure 1CGo shows that supplementation with15 mmol/liter mannitol or 3OMG did not change the IL-12 gene expression, compared with that under NG conditions.


Figure 1
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  		FIG. 1. Effects of HG on mRNA expression of cytokine genes in MPMs. MPMs were treated under HG conditions for the indicated time and total cellular RNA was extracted for real-time RT-PCR analysis. Data were calculated by the 2–{Delta}{Delta}CT method and presented as fold induction of transcript for IL-12 gene normalized to GAPDH in cells treated under HG condition over that under NG condition. Results shown are mean ± SE from three experiments run in triplicate. **, P < 0.01 vs. NG. A, Dose-dependent effects of HG on mRNA expression of cytokine genes in MPMs. B, Time-dependent effects of HG on mRNA expression of cytokine genes in MPMs. C, High osmolality did not induce IL-12 mRNA expression. MPMs were incubated under NG, NG plus 15 mmol/liter 3OMG or 15 mmol/liter mannitol, or NG plus 15 mmol/liter D-glucose (HG) conditions for 3 h. D, IL-12 protein production. MPMs were seeded in 24-well plates with each well containing 1 x 106 cells. Cells were treated with NG or HG for four sequential intervals. The conditioned medium was analyzed for secreted IL-12 protein by ELISA using a specific IL-12 p40 antibody. HG induced IL-12 protein release to the medium at the time interval of 0–16 h. Values are the mean ± SE of triplicate cultures. Results are representative of three experiments. *, P < 0.05 vs. NG; **, P < 0.01 vs. NG; ***, P < 0.001 vs. NG.

 
We next examined whether the HG could alter IL-12 protein levels. MPMs were treated with either NG or HG. Conditioned medium was collected in a sequential fashion at interval of 0–16, 16–24, 24–36, and 36–60 h, with changes of 1 ml fresh medium containing HG at each time interval. Released IL-12 p40 protein was examined using a specific mouse IL-12 p40 immunoassay kit. Figure 1DGo demonstrates that HG significantly induces IL-12 secretion at the interval of 0–16 h. We did not see increases at other intervals (data not shown). Therefore, HG treatment significantly increases both IL-12 mRNA and protein expression.

Enhanced cytokine gene expression in macrophages from STZ-induced diabetic mice and diabetic db/db mice
To test the in vivo effects of diabetes on cytokine expression, macrophages were isolated from either saline-injected control mice or STZ-injected mice. The baseline blood glucose of the mice before getting STZ was 140, 152, 141, and 161 mg/dl (mean = 148.5 ± 5.0). Thioglycollate was injected on d 8 and macrophages were isolated on d 11. The blood glucose of the STZ-induced diabetic mice at the time of euthanasia was 405, 444, 468, and 474 mg/dl (447.7 ± 15.7, P = 0.0002 vs. control mice). Macrophages from saline-injected mice were taken as controls. Thioglycollate injection did not change the value of blood glucose in C57BL6 mice with 141.1 ± 5.6 mg/dl before injection and 154.5 ± 6.4 mg/dl after injection (n = 10, P = 0.135). Figure 2AGo shows that hyperglycemia significantly increases mRNA expression of the cytokine genes of IL-1ß, IL-6, IL-12, IL-18, and TNF{alpha} in MPMs from STZ-induced diabetic mice, compared with that in macrophages from nondiabetic control mice. Noteworthily, a marked, nearly 20-fold and 9-fold increase in IL-12 and IL-1ß mRNA expression could be seen in MPMs from STZ-induced diabetic mice. Macrophages from spontaneous type 2 diabetic db/db (8–10 wk old male) mice were also used for examining the effects of hyperglycemia on cytokine gene expression. In these experiments, macrophages from 8- to 9-wk-old male heterozygotes from same mice colony (db/+) were taken as controls. The blood glucose of the db/+ and db/db mice before being killed was 160 ± 24 and 548 ± 63 mg/dl (P < 0.01 vs. db/+), respectively. Figure 2BGo shows that IL-1ß expression was increased almost 2-fold (P < 0.0001), IL-6 expression was increased about 17-fold (P < 0.0001), IL-12 expression was increased about 9-fold (P < 0.0001), and TNF{alpha} was increased about 1.7-fold (P = 0.0011) in macrophages from db/db mice, compared with that in macrophages from control db/+ mice. These data indicate that hyperglycemia in vivo is a potent stimulus for mRNA expression of inflammatory cytokines in macrophages.


Figure 2
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  		FIG. 2. Enhanced mRNA expression of cytokine genes in STZ-treated diabetic mice and db/db mice. A, STZ or saline was injected ip into four mice each. Thioglycollate was injected ip to control or STZ groups on d 8. MPMs were isolated and total cellular RNA was extracted for real-time RT-PCR analysis as in Fig. 1Go. Results shown are mean ± SE from three experiments run in triplicate. ***, P < 0.001 vs. control. B, Peritoneal macrophages were isolated from diabetic db/db mice and nondiabetic db/+ littermates. Total RNA extracted from these macrophages was subjected to relative RT-PCR analysis using gene-specific primers (for mouse IL-1ß, TNF{alpha}, IL-6, and IL-12) and 18S primers as internal control. PCR products were fractionated on 1.5% agarose gels and photographed using an Alpha-Imager 2000 (Alpha-Imager; Alpha Inotech, San Leandro, CA) documentation and analysis system. Density of DNA bands corresponding to amplified products and 18S RNA was determined with Quantity One software (Bio-Rad). Results are expressed as ratio of specific band to 18S RNA internal standard. Results shown are mean ± SEM from three separate experiments, db/db vs. control is P < 0.001. ***, P < 0.001, db/db vs. control.

 
Role of protein kinase C (PKC) in HG-induced IL-12 mRNA expression
PKC is clearly one of the mechanisms associated with HG-induced complications. We first examined whether HG increased PKC activity in primary cultured macrophages. PKC activation was examined using Western blot with a specific anti-phosphor-PKC (pan) ({gamma}Thr514) antibody and specific antiphosphorylated myristolated alanine-rich C kinase substrate (MARCKS) antibody. Figure 3AGo shows a time-dependent increase in PKC activity, illustrating that {gamma}Thr514 phosphorylated PKC was increased at 5–10 min and a peak at 30 min of HG treatment. The density of phosphorylated MARCKS bands had a similar pattern (Fig. 3BGo), showing an increase at 5–10 min and a peak at 30 min of HG treatment. We then tested whether two structurally distinct PKC inhibitors, GF-109203X or calphostin C attenuated HG effects on IL-12 gene expression. After serum depletion, macrophages were pretreated with fresh RPMI 1640 medium containing GF-109203X (100 nmol/liter), calphostin C (100 nmol/liter), or vehicle control dimethylsulfoxide for 30 min. MPMs were then treated with HG or NG for 3 h. Figure 3CGo shows that GF-109203X or calphostin C completely inhibited HG-induced IL-12 gene expression, suggesting that PKC activation is important in HG effects on IL-12 expression. GF-109203X or calphostin C alone at concentrations studied had no significant effect on IL-12 gene expression under NG condition.


Figure 3
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  		FIG. 3. HG activates PKC activity and the inhibitory effect of PKC inhibitors on HG-induced IL-12 mRNA expression. A, PKC activity was measured by detecting the density changes of phosphorproteins using the Western blot method. Fifty micrograms of macrophage protein were resolved on 10% SDS-polyacrylamide gel and the proteins on the gel were transferred on Immuno-Blot polyvinylidene difluoride membrane and detected by specific phosphor antibody. A, Time course of HG stimulatory effect on the amount of pan ({gamma}Thr514). B, Time course of HG stimulatory effect on the amount of phosphor-MARCKS. C, Inhibitory effects of PKC inhibitors on HG-induced IL-12 mRNA expression. MPMs were pretreated with calphostin C (100 nmol/liter) or GF-109203X (100 nmol/liter) in NG medium for 30 min. The cells were then incubated in NG or HG media for an additional 6 h. Total cellular RNA was extracted for real-time RT-PCR analysis as in Fig. 1Go. Results are mean ± SE of three independent experiments run in triplicate. **, P < 0.01 vs. NG; Figure 3Figure 3, P < 0.01 vs. HG.

 
Role of p38 MAPK in HG-induced IL-12 expression
Figure 4AGo shows a time course of p38 MAPK activity detected by Western blot using a specific anti-phospho-p38 antibody. HG-induced p38 activity occurred as early as the 5-min treatment period and reached a peak at 10 min. ß-Actin on the stripped membrane was detected with anti-ß-actin antibody, which showed equal protein loading among different samples. We also tested the effect of the relatively specific p38 MAPK inhibitor, SB 202190. After serum depletion, macrophages were pretreated with fresh RPMI 1640 medium containing either 2 µmol/liter SB 202190 or dimethylsulfoxide vehicle for 30 min. MPMs were then treated with HG or NG for 3 h. Figure 4BGo shows that even though SB 202190 alone had a slight effect to increase basal IL-12 gene expression in NG, SB 202190 almost completely inhibited HG-induced IL-12 gene expression. These results suggest that p38 MAPK is also important for HG action.


Figure 4
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  		FIG. 4. HG activates p38 kinase and effect of p38 kinase inhibitor on HG-induced IL-12 mRNA expression. A, Time course of HG stimulatory effects on phosphor-p38 protein detected by Western blot. The Western blot conditions were the same as in Fig. 3AGo. B, Inhibitory effects of p38 inhibitor on HG-induced IL-12 mRNA expression. MPMs were pretreated with either SB 202190 (2 µmol/liter) or vehicle in NG or HG medium for 30 min. The cells were then incubated in NG or HG media for an additional 3 h. Total cellular RNA was extracted for real-time RT-PCR analysis as in Fig. 1Go. Results are mean ± SE of three independent experiments run in triplicate. *, P < 0.05 vs. NG; ¤, P < 0.05 vs. HG.

 
Role of JNK in HG-induced IL-12 gene expression
JNK has been implicated in the cascade of glucose-induced complications. Figure 5AGo shows that HG activates JNK in cultured macrophages. To explore whether the JNK pathway is involved in HG action on IL-12, we tested the effect of the specific JNK inhibitor, JNKI-1, a peptide that competitively blocks the interaction between JNK and c-Jun. After depletion, macrophages were pretreated with fresh RPMI 1640 medium containing 10 µmol/liter JNKI-1 or 10 µmol/liter control peptide for 30 min, and MPMs were then treated with HG or NG for 3 h. Figure 5BGo shows that JNKI-1 significantly inhibited HG-induced IL-12 gene expression, whereas the JNKI control peptide had no effect. Neither the JNKI nor the JNKI control peptide had an effect on IL-12 mRNA expression under NG conditions. These data suggest that the JNK pathway is involved in HG-induced IL-12 mRNA expression.


Figure 5
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  		FIG. 5. HG activates JNK activity and effect of JNK inhibitor on HG-induced IL-12 mRNA expression. A, Time course of HG stimulatory effects on phosphor-JNK protein detected by Western blot. The Western blot conditions were the same as in Fig. 3AGo. B, Inhibitory effects of JNK inhibitor on HG-induced IL-12 mRNA expression. MPMs were pretreated with or without JNKI-1 (10 µmol/liter) or its control peptide (10 µmol/liter) in NG or HG medium for 30 min. The cells were then incubated in NG or HG media for an additional 3 h. Total cellular RNA was extracted for real-time RT-PCR analysis as in Fig. 1Go. Results are mean ± SE of three experiments run in triplicate. **, P < 0.01 vs. NG; ¤¤, P < 0.01 vs. HG.

 
NF{kappa}B is also central to glucose-induced complications and oxidative stress. Because the IKK complex appears to be critical for NF{kappa}B activation, IKK was used as an indicator of NF{kappa}B activation. Figure 6AGo shows that HG time-dependently activated IKK activity. To explore the role of the NF{kappa}B pathway in our model, we tested whether an NF{kappa}B inhibitor could attenuate HG effects on IL-12 gene expression. After serum depletion, macrophages were pretreated with fresh RPMI 1640 medium containing 75 µg/ml of a specific NF{kappa}B inhibitor or 75 µg/ml control NF{kappa}B inhibitor (inactive control for NF{kappa}B inhibitor) for 30 min. MPMs were then treated with HG or NG for 3 h. Figure 6BGo shows that the active NF{kappa}B inhibitor completely blocked HG-induced IL-12 gene expression, whereas the NF{kappa}B inhibitor control had no effect. Neither the NF{kappa}B inhibitor nor the NF{kappa}B control compound altered IL-12 mRNA expression under NG conditions. These data suggest that the NF{kappa}B pathway is implicated HG action on IL-12 mRNA expression.


Figure 6
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  		FIG. 6. HG activates IKK activity and effect of NF{kappa}B inhibitor on HG-induced IL-12 mRNA expression. A, Time course of HG stimulatory effects on phosphor-IKK protein detected by Western blot. The Western blot conditions were the same as in Fig. 3AGo. B, Inhibitory effects of NF{kappa}B inhibitor on HG-induced IL-12 mRNA expression. MPMs were pretreated with NF{kappa}B inhibitor 1 or its control peptide (75 µg/ml) in NG or HG medium for 30 min. The cells were then incubated in NG or HG media for an additional 3 h. Total cellular RNA was extracted for real-time RT-PCR analysis as in Fig. 1Go. Results are mean ± SE of three experiments run in triplicate. **, P < 0.01 vs. NG; ¤¤, P < 0.01 vs. HG.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we investigated the effect of HG on the mRNA and protein expression of inflammatory cytokines. This study demonstrates that high glucose induces a significant increase in the mRNA expression of several important inflammatory cytokines including IL-1ß, IL-6, IL-12, and TNF{alpha}. Taking IL-12 as an example, we have demonstrated that the HG-induced increase of mRNA expression was not elicited by high osmotic pressure because 25 mmol/liter concentration of D-glucose + mannitol or of D-glucose + 3OMG did not change the mRNA expression of IL-12. We also demonstrate that HG induced an increase in IL-12 protein secretion from macrophages. This is the first observation showing that HG activates mRNA expression of multiple cytokines in primary cultured MPMs. We further demonstrate that hyperglycemia increases the mRNA expression of multiple cytokine genes in macrophages isolated from STZ-induced diabetic mice as well as type 2 diabetic db/db mice, showing that the stimulatory effects of hyperglycemia on cytokine gene expression exists in vivo and suggesting that glucose independent of autoimmunity can activate these important cytokines.

Activation of IL-12 and downstream transcription factors have been linked to atherosclerosis in mouse models and humans (6, 7, 8). Therefore, IL-12 was taken as an example to further investigate the regulatory mechanism of HG stimulation on cytokine mRNA expression.

It has been well established that increasing extracellular glucose concentration from 5.6 to 22.2 mmol/liter appears sufficient to accomplish an activation of diacylglycerol (DAG) and PKC (13). DAG-PKC activation has been considered an independent biochemical pathway involved in the pathogenesis of diabetic complications and is involved in the regulation of various cell functions including cytokine actions (14). There was a report showing an involvement of PKC in HG-induced MCP-1 in human peritoneal mesothelial cells (15). Interestingly, MCP-1 is one of the target genes produced by downstream transcription factor activation by IL-12. We first demonstrated that HG activated PKC activity by detecting an increase in the amount of phospho-PKC (pan) at {gamma}Thr514 using specific anti-pan {gamma}Thr51 antibody. It can be seen that HG time-dependently increases the density of pan. MARCKS protein is a widely accepted indicator of PKC activation. The level of phosphorylated MARCKS is generally believed to reflect PKC activation. Our data clearly illustrated that HG increases the density of phosphorylated MARCKS protein and the increase of the density of phosphor-MARCK protein has a same time pattern with that of the density changes of pan protein, strongly indicating that HG activates PKC activity in primary cultured macrophages. To investigate the possible involvement of PKC in HG-induced increases in IL-12 mRNA expression, two specific and structurally distinct PKC inhibitors, GF 109230X and calphostin C, were used. Both inhibitors completely blocked HG-induced IL-12 mRNA expression, strongly suggesting a role of DAG-PKC activation in HG-induced IL-12 mRNA expression. The PKC family includes at least 11 isoforms. There are reports showing that macrophages from various tissues express multiple types of PKC isoforms including {alpha}, ß1 and 2, {gamma}, {epsilon}, {eta}, and {zeta}. However, it has been hypothesized that macrophage expression/function of PKC isoforms is tissue specific (alveolar vs. peritoneal) (16). Further studies will be necessary to elucidate the types of PKC isoforms that MPMs express and which isoform(s) of PKC mediate HG-induced IL-12 mRNA expression in murine peritoneal macrophages.

There is increasing evidence demonstrating a critical role of MAPK activation in the inflammatory response of macrophages (17). Evidence also shows that MAPK phosphatase-1, which dephosphorylates and inactivates these MAPKs, plays an important role in cytokine production induced by agonists. Stimulation of primary macrophages isolated from MAPK phosphatase-1-deficient mice by lipopolysaccharide resulted in a prolonged p38 MAPK phosphorylationand more robust and rapid TNF{alpha} production, compared with wild-type macrophages (18). In the present study, we not only demonstrated that HG activated p38 activity in macrophages, which is consistent with other reports, but also a p38 MAPK inhibitor, SB202190, significantly reduced HG-induced IL-12 mRNA expression, suggesting an involvement of the p38 MAPK pathway. Our data are consistent with the data in a report showing that the inhibition of the phosphorylation of p38 MAPK is concomitant with a down-regulation of IL-12 p40 production (19).

JNK is an inflammation-related MAPK (20). In the present study, we used JNKI-1 as a relatively specific inhibitor for JNK-1, JNK-2, and JNK-3. JNKI-1 works by inhibiting the interaction between JNK and its substrate (21). JNKI-1 almost completely suppressed HG-induced IL-12 mRNA expression without effect on basal IL-12 mRNA expression. In contrast, the JNK inhibitor 1 control peptide, an inactive analog of JNKI-1, did not block HG-induced IL-12 mRNA expression, further supporting the conclusion that JNKI-1 specifically inhibited and JNK mediated HG-induced IL-12 mRNA expression.

JNK has been directly linked to NF{kappa}B regulation (22). Furthermore, evidence shows that IL-12 is an inducible, heterodimeric, disulfide-linked cytokine composed of p35 and p40 subunits. Expression of the p35 subunit is constitutive and ubiquitous (23), whereas the biological activity of IL-12 is regulated mainly by the induction of p40, which reported to be regulated primarily at the level of transcription including NF{kappa}B (24). For NF{kappa}B activation, phosphorylation of IKK appears to be critical. Therefore, we chose the phosphorylation of IKK as an indicator of NF{kappa}B activation. Our data clearly show an increase in the amount of phosphorylated IKK in HG conditions vs. NG condition, demonstrating an stimulatory effect of HG on NF{kappa}B activation. NF{kappa}B inhibitor, which contains the nuclear localization sequence residues 360–369 of NF{kappa}B p50 and inhibits translocation of the NF{kappa}B active complex into the nucleus (25), was used for evaluating whether NF{kappa}B is involved in the regulation of HG-induced IL-12 mRNA expression. Our results demonstrate that blockade of the NF{kappa}B translocation completely suppresses HG-induced IL-12 mRNA expression, strongly suggesting an involvement of NF{kappa}B in the action of glucose.

We have not pursued in this study whether there is a signaling linearity connecting PKC, p38 MAPK, JNK, and NF{kappa}B. Data have shown that pretreatment of neutrophils with the structurally distinct PKC{alpha}/ß inhibitors Go6976 or GF109203X decreased nuclear translocation of NF{kappa}B and production of the proinflammatory cytokine TNF{alpha}. These inhibitors also prevented lipopolysaccharide-induced phosphorylation of IKK{alpha}/ß, phosphorylation and degradation of I{kappa}B-{alpha}, and phosphorylation of the 65 subunit of NF{kappa}B. Activation of p38 and JNK also was diminished in neutrophils in which PKC{alpha}/ß was inhibited (26). These data clearly suggest a cascade involving PKC to MAPK to NF{kappa}B to cytokine production in neutrophils. The signal transduction linking PKC, MAPK, and NF{kappa}B in HG-induced IL-12 mRNA expression will require further investigation. However, elegant studies have shown that a role for mitochondrial-induced oxidative stress as a common pathway leading to glucose-induced complications (14). It will be interesting in future studies to explore the role of the mitochondria in glucose-induced IL-12 production in macrophages. Our studies also are supported by a recent report (27) showing a key role of oxidative stress in generating IL-12 in mouse peritoneal macrophages.

Summary
In the present study, HG induced an increase in multiple proinflammatory cytokines in MPMs, in vitro and in two diabetic mouse models. This increase induced by HG was not due to osmotic pressure, suggesting that this is a specific effect by high glucose. IL-12 has been taken as an example, and our data show that PKC, p38 MAPK, JNK, and NF{kappa}B are involved in the regulation of HG-induced IL-12 gene expression. These results suggest that glucose-induced macrophage generation of proinflammatory could be involved in both short-term and long-term vascular complication of diabetes.


   Acknowledgments
 
The authors express their deep thanks to Micah Nadler for preparing the figures and Mr. George Vandehoff and Jessica Kurdica for their technical assistance.


   Footnotes
 
This work was supported by National Institutes of Health Grants P01 HL 55798 and RO1 DK 55240 and the American Diabetes Association.

The authors have nothing to declare.

First Published Online February 2, 2006

Abbreviations: DAG, Diacylglycerol; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HG, high glucose; IKK, inhibitory-{kappa}B kinase; JNK, c-jun-N-terminal kinase; MCP, monocyte chemoattractant protein; MPM, mouse peritoneal macrophage; NF{kappa}B, nuclear factor-{kappa}B; NG, normal glucose; 3OMG, 3-O-methyl-glucose; p38, p38 MAPK; pan, phosphor-PKC; PKC, protein kinase C; STZ, streptozotocin.

Received April 29, 2005.

Accepted for publication January 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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