Expression of the inflammatory cytokine IL-1β occurs in various inflammatory diseases, and IL-1β production is regulated at multiple levels. There are conflicting reports about the effects of antioxidants on IL-1β production. In this study, we investigated the regulatory role of the antioxidant DMSO on LPS-stimulated IL-1β gene expression in human PBMC and in vivo. This study demonstrated that 1% DMSO increased LPS-stimulated (50 ng/ml) IL-1β secretion in a dose- and time-dependent manner without altering TNF or IL-6. DMSO also elevated IL-1β secretion by PBMC in response to exogenous superoxide anions. Despite the increase in IL-1β, there was no augmentation of NF-κB with the addition of DMSO. The steady state mRNA coding for IL-1β following LPS stimulation was also increased. Cycloheximide studies demonstrated that the DMSO augmentation of IL-1β mRNA did not require de novo protein synthesis, and studies with actinomycin D showed that DMSO did not alter the half-life of IL-1β mRNA, suggesting that DMSO did not change the stability of IL-1β mRNA. Experiments using a reporter vector containing the 5′-flanking region of the human IL-1β gene revealed that DMSO augmented LPS-induced IL-1β reporter activity. In vivo, treatment of mice with DMSO significantly increased plasma levels of IL-1β after endotoxin challenge. These data indicate that DMSO directly increases LPS-stimulated IL-1β protein production through the mechanisms of augmenting promoter activity and increasing mRNA levels.

The IL-1β cytokine, involved in several inflammatory and immunologic processes, is produced by activated monocytes/macrophages, fibroblasts, endothelial cells, as well as many other cell types (1). IL-1 represents a potent inflammatory cytokine with numerous biologic activities that regulate host defense and immune responses (2). A precursor form of IL-1β, referred to as pro-IL-1β, is first translated in the cytosol of macrophages. Pro-IL-1β is a 31–34-kDa nonactive form of the cytokine that is subsequently cleaved via an enzymatic process into the 17-kDa, mature functional form by IL-1 converting enzyme (ICE) 3 or caspase 1 (3, 4, 5, 6). After the ICE/caspase cleavage, the released, active IL-1 exhibits diverse biologic functions. Within the peripheral blood, monocytes are the main source of IL-1β in response to LPS (7).

Bacterial LPS, a major component of the outer membrane of Gram-negative bacteria, contributes to the local and systemic toxicity of Gram-negative infections (8). It is a potent activator of the immune system that induces local inflammation, Ab production, and shock when infused at high concentrations (9). Monocytes play a central role in host defense against bacterial infection and are major cellular targets for LPS action. LPS regulates several macrophage functions (10, 11) including the induction of inflammatory cytokines such as IL-1, IL-6, and TNF (12, 13, 14, 15). These cytokines subsequently exert pleiotropic biologic effects on a wide range of target cells (1, 16, 17).

After LPS interacts with the TLR4 complex, multistep pathways are triggered resulting in IL-1β production by monocytes and macrophages. Although many of the steps of the pathways have been elucidated, several aspects remain to be defined (18). Both direct and indirect evidence has shown that monocytes/macrophages produce reactive oxygen intermediates (ROI) in response to LPS. Further, these endogenously produced ROI participate in the LPS-induced inflammatory mediator production by monocytes/macrophages (19). However, there are conflicting reports about the effect of antioxidants on IL-1β production. Previous investigators have reported that treatment with antioxidant reagents inhibited LPS-induced IL-1β production by monocytes/macrophages (20, 21, 22, 23). In contrast, we found that the antioxidant DMSO, a scavenger of the hydroxyl radical (OH), augmented IL-1β production in LPS-stimulated whole blood (24, 25).

DMSO [(CH3)2SO] is an amphipathic molecule with a highly polar domain and two apolar groups, making it soluble in both aqueous and organic media. It has been used successfully in the treatment of dermatological, urinary, pulmonary, rheumatic, and renal manifestations of amyloidosis. Due to its anti-inflammatory properties and the ability to scavenge reactive oxygen species, DMSO has been purposed for the treatment of several diseases (reviewed in Ref. 26).

In this report, we investigated the DMSO regulation of LPS-stimulated IL-1β production in isolated human PBMC. We sought to determine whether DMSO will augment IL-1β, and whether this regulation occurs at the transcriptional or translational level.

LPS (Escherichia coli serotype O111:B4), xanthine, xanthine oxidase, DMSO, 3,3′,5,5′-tetramethylbenzidine, N-acetyl-l-cysteine (NAC), and dimethylthiourea (DMTU) were obtained from Sigma-Aldrich. Capture and biotin-conjugated detection Abs for ELISA measurement of human and mouse IL-1β, TNF-α, and IL-6, were purchased from R&D Systems. A complete kit for the determination of pro-IL-1β was also purchased from R&D Systems. HRP-conjugated streptavidin was purchased from Jackson ImmunoResearch Laboratories. The blocking solutions, blotto and casein, and T-PER Tissue Protein Extraction Reagent were obtained from Pierce. NycoPrep (1.077 ± 0.002 g/ml) and RPMI 1640 were obtained from Invitrogen Life Technologies. The in vitro transcription kit and the RPA kit were obtained from BD Pharmingen. [α-32P]UTP was purchased from New England Nuclear. The Lipofectamine Plus transfection kit and the Zero Blunt TOPO PCR Cloning kit were from Invitrogen Life Technologies. The HL-14 template set was the generous gift of Dr. R. Rochford (Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI). The HL-14 template set synthesized riboprobes specific for the mRNA of IL-6, IL-10, IL-1α, TNF-β, GM-CSF, TGF-β 1, IL-1β, TNF-α, and L32. The Dual Luciferase Reporter Assay System, pGL-3 basic vector, pRL-TK vector, Wizard Genomic DNA Purification kit, Wizard Plus SV Miniprep DNA purification system, and restriction enzymes were from Promega. The endoFree plasmid maxi kit was from Qiagen, and the expand high fidelity PCR System was from Roche.

PBMC were isolated from the peripheral blood of healthy volunteers by a density gradient centrifugation. Freshly drawn, heparinized whole blood was gently mixed with one volume of RPMI 1640. The diluted blood was layered over NycoPrep (Nycomed) and centrifuged at 800 × g, 4°C for 20 min. PBMC were collected from the interface between the plasma and the density gradient solution. After washing in PBS, PBMC were resuspended in RPMI 1640 containing 5% autologous plasma at a final cell concentration of 106/ml. These studies have been approved by the Institutional Review Board of the University of Michigan.

PBMC were cultured in 1 ml volumes in 24-well cell culture plates at 37°C with 5% CO2. The cells were cultured with or without different concentrations of LPS, DMSO, NAC, DMTU, or xanthine/xanthine oxidase. Cell-free supernatants were harvested after 24 h of culture or at varying time points (as described in the kinetics experiments), centrifuged at 1500 × g for 10 min, and stored at −20°C.

Cells from the murine macrophage cell line, RAW 264.7 (American Type Culture Collection) were maintained in DMEM containing 1 mM sodium pyruvate, 2 mM glutamine, 100 U/ml penicillin/streptomycin, and 10% FBS.

Female ICR mice (25–30 g) were obtained from Harlan Sprague Dawley and maintained under standard laboratory conditions. All experiments were performed in accordance with the National Institutes of Health guidelines and approved by the University of Michigan Animal Use Committee. DMSO (1.5 ml/kg) was given i.p. 10 min before an i.p. injection of 10 μg/mouse of LPS. Control mice received normal saline as a vehicle. Blood was collected by cardiac puncture 2 h after LPS injection, and plasma was collected for cytokine detection.

Culture supernatants from the stimulated mononuclear cells were isolated, and human IL-1β, TNF-α, and IL-6 levels were determined by ELISA according to our previously published methods (27), except for intracellular pro-IL-1β, which was detected by using the pro-IL-1β ELISA kit. Mouse IL-1β was detected by ELISA using a similar protocol. After the culture supernatants were removed, the cells were washed with PBS and lysed using 200 μl of T-PER Tissue Protein Extraction Reagent. The cell lysates were subjected to ELISA for detecting pro-IL-1β following the manufacturer’s instructions.

Clean instruments were used for each cell sample to eliminate cross-contamination. Total RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform extraction procedure (28). Briefly, the cells were homogenized in guanidinium thiocyanate followed by acid phenol-chloroform extraction and then precipitated with isopropanol.

The RPA was performed following the manufacturer’s instructions (BD Pharmingen). Riboprobe syntheses was driven by the T7 bacteriophage RNA polymerase with [α-32P]UTP as the labeling nucleotide. 32P probe (6 × 105 cpm/sample) was added to tubes containing the sample RNA (4 μg), which was dissolved in 8 μl of hybridization buffer. The samples were heated to 90°C, the temperature slowly decreased to 56°C, and then incubated overnight. The dsRNA was then digested by RNase A and RNase T1 followed by treatment with proteinase K. The RNA duplexes were isolated by extraction/precipitation as discussed, dissolved in 5 μl of gel loading buffer, and electrophoresed in standard 5% acrylamide/8 M urea sequencing gels. The gel was dried and placed on XAR film (Eastman Kodak) with intensifying screens and was developed at −70°C for 4–12 h.

Analysis of the specific binding of p65/p50 to their DNA consensus oligonucleotides was performed in nuclear extracts using the ELISA-based transcription factor assay (all reagents from Active Motif). Human PBMC in 24-well culture plates were treated with 50 ng/ml LPS in the presence or absence of 1% DMSO. Cells were collected, washed two times in PBS, and then nuclear extracts were obtained using the Nuclear Extract kit. The activity of p50 and p65 NF-κB was assayed using the TransAM NF-κB kit following the manufacturer’s instructions. The assay plate has an immobilized oligonucleotide containing the NF-κB consensus site (5′-GGGACTTTCC-3′). The active form of NF-κB contained in nuclear extracts specifically binds to this oligonucleotide. The primary Ab used to detect NF-κB recognizes an epitope on p50 or p65 that is accessible only when NF-κB p50 and p65 are bound to the target DNA. The level of nuclear NF-κB p50 and p65 was expressed in the OD value.

The pGL-3 basic vector was used to construct the pGL-3 basic-human IL-1β promoter region reporter vector (pGL-3-IL-1β). The sequence of human IL-1β gene (X04500) (29, 30) and the promoter (U26540) (31) were obtained from National Center for Biotechnology Information (〈www.ncbi.nlm.nih.gov/〉). The known promoter region of human IL-1β (from −6741 to +42) was amplified by using the Expand High Fidelity PCR System with primers and genomic DNA from human white cells as the template. The up-stream primer was 5′-ACGCGTTACAATGTCAATGCCAGT-3′ corresponding to nucleotides −6741 to −6718 of the sense strand. The up-stream primer contains the MluI restriction site at the 5′ end. The down-stream primer was 5′-GCTAGCAGCCTGTTGTGCCTTG-3′ corresponding to +21 to +42. The downstream primer contains the NheI restriction site at the 5′ end. The PCR fragment was cloned into the Zero Blunt TOPO PCR Cloning vector. The inserted positive vector was digested with MluI and NheI, then the IL-1β promoter fragment was subcloned into the MluI-NheI site in the pGL-3 basic vector to generate the reporter vector. The cloned 5′-flanking region sequence of IL-1β gene in reporter vector was confirmed by sequence analysis performed at University of Michigan DNA sequencing core (Ann Arbor, MI).

Transient cotransfection of the pGL-3 reporter vector and the control vector pRL-TK into RAW 264.7 cells was performed using the Lipofectamine Plus method. The total amount of DNA was 0.4 μg/well. The molar ratio of pGL-3 reporter vector to control vector pRL-TK vector was 10:1. The DNA complexes were made and the RAW 264.7 cells were transfected following the manufacturer’s instructions.

After incubation with the DNA complexes for 6 h, the cells were washed twice with warm, fresh DMEM and DMEM growth medium was added to the cells. DMSO and LPS were added to the final concentrations of 1% and 10 μg/ml, respectively. The final volume of medium was 1 ml. After 12 h of stimulation, the cells were washed twice with PBS (without Ca2+ and Mg2+), and 100 μl of 5× passive lysis buffer (dual luciferase reporter assay system) was added to each well. The plate was agitated for 15 min at room temperature and then stored at −70°C.

After preparation of the cell extracts, luciferase activities were measured using the dual luciferase reporter assay system with a Packard LumiCount BL10000 (Packard Instrument) per the manufacturer’s instructions. The activities of the reporter vectors were reported as the activity ratio of firefly luciferase to the control vector Renilla luciferase.

All analyses were performed with GraphPad Prism version 4.00 for Windows (GraphPad Software; 〈www.graphpad.com〉). Results were reported as the mean ± SEM. Statistical comparisons were made using a one-way ANOVA with the Turkey-Kramer multiple comparison tests. For direct comparisons between groups, t tests were used.

We previously reported that DMSO augmented LPS-stimulated IL-1β production in whole blood (25). Because monocytes are the principal source of most proinflammatory cytokines in blood (7), we evaluated isolated PBMC in this study. The PBMC were treated with DMSO (1%, v/v) immediately before LPS stimulation to analyze whether DMSO would increase or decrease proinflammatory cytokine release. DMSO alone, i.e., without LPS stimulation, had no effect on IL-1β, TNF-α, or IL-6 release from PBMC (data not shown). Unstimulated secretion of IL-1β was nearly undetectable and LPS stimulation resulted in the secretion of IL-1β into the tissue culture supernatant. DMSO increased the LPS-induced secretion of IL-1β by nearly 2-fold (Fig. 1). To identify whether this was a nonspecific effect such as increased cell viability, we measured two other proinflammatory cytokines, TNF-α and IL-6. DMSO neither suppressed nor elevated the LPS induced levels of TNF-α or IL-6 (Fig. 1, B and C). To further test the role of antioxidants in the regulation of IL-1β, two additional antioxidants, NAC and DMTU, were examined. Treatment with 10 mM NAC increased LPS-stimulated IL-1β production from 4402 ± 974 to 8310 ± 1589 pg/ml (n = 6, p < 0.01). In a similar manner, treatment with 10 mM DMTU resulted in an increase of LPS-stimulated IL-1β from 2454 ± 400 to 6334 ± 938 pg/ml (n = 6, p < 0.01).

FIGURE 1.

Effect of DMSO on LPS-induced cytokine production by human PBMC. Control represents no LPS stimulation. IL-1β (A), TNF-α (B), and IL-6 (C) are represented. The PBMC were pretreated with DMSO (1%, v/v) immediately prior LPS (50 ng/ml) stimulation and culture supernatants were collected after 24 h. Data were presented as mean ± SEM (n = 31). ∗, p < 0.01 vs control; #, p < 0.01 vs LPS stimulation alone.

FIGURE 1.

Effect of DMSO on LPS-induced cytokine production by human PBMC. Control represents no LPS stimulation. IL-1β (A), TNF-α (B), and IL-6 (C) are represented. The PBMC were pretreated with DMSO (1%, v/v) immediately prior LPS (50 ng/ml) stimulation and culture supernatants were collected after 24 h. Data were presented as mean ± SEM (n = 31). ∗, p < 0.01 vs control; #, p < 0.01 vs LPS stimulation alone.

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To further investigate DMSO augmentation of LPS-induced IL-1β production, the effects of DMSO at several time points after LPS stimulation were examined. Specifically, the concentration of IL-1β after stimulation with 50 ng/ml LPS with and without 1% DMSO was examined over a 24-h time period. After 2 h, IL-1β increased rapidly until the 8-h time point (2150 ± 500 pg/ml) and thereafter reached a plateau (Fig. 2 A). In the presence of 1% DMSO, the resulting kinetic curve was similar to the curve without DMSO, but the levels of IL-1β were increased nearly 2-fold.

FIGURE 2.

Kinetics and dose response of DMSO augmentation of IL-1β. A, Kinetics of IL-1β production stimulated by LPS (50 ng/ml) in the presence or absence of DMSO. PBMC were stimulated with 50 ng/ml LPS and the cell culture supernatants tested for IL-1β. Data are presented as mean ± SEM (n = 5–8). #, p < 0.01 vs LPS stimulation alone. B, Effect of DMSO on LPS-induced dose response of IL-1β production by PBMC. PBMC were stimulated with the indicated concentrations of LPS in the presence or absence of DMSO (1.0%) for 24 h. Data were presented as mean ± SEM (n = 5–8). #, p < 0.01 vs LPS stimulation alone.

FIGURE 2.

Kinetics and dose response of DMSO augmentation of IL-1β. A, Kinetics of IL-1β production stimulated by LPS (50 ng/ml) in the presence or absence of DMSO. PBMC were stimulated with 50 ng/ml LPS and the cell culture supernatants tested for IL-1β. Data are presented as mean ± SEM (n = 5–8). #, p < 0.01 vs LPS stimulation alone. B, Effect of DMSO on LPS-induced dose response of IL-1β production by PBMC. PBMC were stimulated with the indicated concentrations of LPS in the presence or absence of DMSO (1.0%) for 24 h. Data were presented as mean ± SEM (n = 5–8). #, p < 0.01 vs LPS stimulation alone.

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The kinetic studies were performed using an LPS concentration of 50 ng/ml. The next studies were performed to determine whether the DMSO augmentation would occur over a wide range of LPS concentrations. LPS alone induced near maximal IL-1β production at a concentration of 0.5 ng/ml, and DMSO caused a significant increase in IL-1β production at every LPS concentration above 0.05 ng/ml (Fig. 2 B). There was no significant change in either TNF or IL-6 production (data not shown).

As shown earlier, DMSO augmented LPS-induced IL-1β release by PBMC in a time- and dose-dependent manner. There are multiple levels at which the regulation could occur, including increasing the amount of the intracellular precursor protein pro-IL-1β. PBMC were incubated for 24 h with LPS (50 ng/ml) in the presence of DMSO, and supernatant levels of IL-1β and intracellular pro-IL-1β levels were measured. Consistent with Fig. 1, DMSO augmented the LPS-induced IL-1β release in the culture supernatants (Fig. 3,A). In contrast to the extracellular increases in IL-1β, the levels of intracellular pro-IL-1β were slightly decreased by treatment with DMSO although this reduction was not significant (Fig. 3 B).

FIGURE 3.

DMSO effects on pro-IL-1β. PBMC were treated with DMSO immediately before stimulation with 50 ng/ml LPS. A, Extracellular mature IL-1β demonstrating that DMSO augments extracellular supernatant IL-1β. B, Intracellular pro-IL-1β is demonstrated. There was a slight decrease in the pro-IL-1β but it was not significant. Data were presented as mean ± SEM (n = 5). ∗, p < 0.01 vs control.

FIGURE 3.

DMSO effects on pro-IL-1β. PBMC were treated with DMSO immediately before stimulation with 50 ng/ml LPS. A, Extracellular mature IL-1β demonstrating that DMSO augments extracellular supernatant IL-1β. B, Intracellular pro-IL-1β is demonstrated. There was a slight decrease in the pro-IL-1β but it was not significant. Data were presented as mean ± SEM (n = 5). ∗, p < 0.01 vs control.

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It has been reported that antioxidant reagents inhibit LPS-induced IL-1β, TNF-α, and IL-6 production by monocytes/macrophages (20, 32, 33). Koga et al. (34) reported that hydroxyl free radical scavengers inhibit IL-1β production induced by reoxygenation, and that oxygen free radicals are capable of triggering IL-1β production by monocytes. However, in contrast to these reports, DMSO significantly increased the LPS-induced IL-1β production in our studies. This suggests that DMSO did not work solely as a free radical scavenger in augmenting IL-1β production. To more precisely map the actions of DMSO, PBMC were treated with an generating system consisting of xanthine (200 μM) plus xanthine oxidase (10 mU/ml) for 24 h in the presence or absence of 1% DMSO. The previous data shows that scavenging of ROI would augment IL-1β production when cells have been stimulated with LPS. These experiments would determine whether the addition of exogenous oxidants would induce cytokine production, and whether DMSO would further increase IL-1β. As anticipated, xanthine/xanthine oxidase induced IL-1β production in the PBMC. The addition of DMSO further increased IL-1β production (Fig. 4,A). Xanthine/xanthine oxidase also induced TNF-α and IL-6 production above unstimulated levels, but DMSO did not further increase the levels of these cytokines in the supernatant (Fig. 4, B and C). These data demonstrate that DMSO will specifically increase IL-1β production whether induced by LPS or exogenous oxidants.

FIGURE 4.

Effects of superoxide anions and DMSO on cytokine production. Cells were cultured in the presence or absence of xanthine/xanthine oxidase (X+XO, 10 mU/ml) for 24 h. Superoxide anions induce IL-1β, TNF-α, and IL-6, but DMSO only augmented IL-1β production. IL-1β (A), TNF-α (B), and IL-6 (C) are represented. Data were presented as mean ± SEM (n = 5). ∗, p < 0.01 vs control; #, p < 0.01 DMSO compared with xanthine/xanthine oxidase alone.

FIGURE 4.

Effects of superoxide anions and DMSO on cytokine production. Cells were cultured in the presence or absence of xanthine/xanthine oxidase (X+XO, 10 mU/ml) for 24 h. Superoxide anions induce IL-1β, TNF-α, and IL-6, but DMSO only augmented IL-1β production. IL-1β (A), TNF-α (B), and IL-6 (C) are represented. Data were presented as mean ± SEM (n = 5). ∗, p < 0.01 vs control; #, p < 0.01 DMSO compared with xanthine/xanthine oxidase alone.

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NF-κB is an important transcription factor in cellular regulation and has been shown to be critical for the induction of IL-1β response to LPS (35). Human PBMC were stimulated with 50 ng/ml LPS for 15, 45, and 90 min in the presence or absence of 1% DMSO. Nuclear extracts were assayed for the effect of DMSO on the LPS-induced increase in the specific binding of p65 and p50 to the consensus NF-κB site. As shown in Fig. 5, A and B, nuclear translocation of NF-κB was significantly increased in PBMC stimulated with LPS compared with that seen in unstimulated cells. DMSO did not alter this LPS-induced increase in the nuclear translocation of NF-κB at any of the time points examined. Thus, the mechanism of the DMSO augmentation of IL-1β was not mediated through these nuclear transcription factors.

FIGURE 5.

Effect of DMSO on LPS-induced activity of NF-κB in PBMC. PBMC were preincubated with DMSO (1.0%) immediately before LPS stimulation. LPS (50 ng/ml) was added, and NF-κB binding examined. Control indicates no LPS stimulation. Specific binding of p50 and p65 to DNA was analyzed in the nuclear extract (2 μg) using an ELISA based technique. A, Specific p50 binding to DNA. B, Specific p65 binding to DNA. Binding activity was expressed as OD values. Although NF-κB was increased with LPS stimulation, there was no augmentation with DMSO. Data are expressed as mean ± SEM (n = 5).

FIGURE 5.

Effect of DMSO on LPS-induced activity of NF-κB in PBMC. PBMC were preincubated with DMSO (1.0%) immediately before LPS stimulation. LPS (50 ng/ml) was added, and NF-κB binding examined. Control indicates no LPS stimulation. Specific binding of p50 and p65 to DNA was analyzed in the nuclear extract (2 μg) using an ELISA based technique. A, Specific p50 binding to DNA. B, Specific p65 binding to DNA. Binding activity was expressed as OD values. Although NF-κB was increased with LPS stimulation, there was no augmentation with DMSO. Data are expressed as mean ± SEM (n = 5).

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Because DMSO enhanced LPS-induced IL-1β production, we investigated the level of this regulation by examining mRNA expression in response to graded doses of DMSO (0.5, 1%). These experiments would determine whether the increases of IL-1β production were due to augmented mRNA levels. IL-1β mRNA expression after 3 h of LPS stimulation was measured using the HL-14 template set of synthesized riboprobes specific for IL-6, IL-10, IL-1α, TNF-β, GM-CSF, TGFβ 1, IL-1β, TNF-α, and L32 (Fig. 6). The graphed data (Fig. 6,B) represent the compiled results from the RPA of three individuals. As shown in Fig. 6 A, different concentrations of DMSO did not alter the level of the housekeeping gene L32, indicating that treatment with DMSO did not increase the viability of PBMC. DMSO alone did not induce IL-1β mRNA. Stimulation with 50 ng/ml LPS induced significant increases in IL-1β, IL-1α, IL-6, and TNF-α, as expected. Consistent with the cytokine protein levels, DMSO specifically increased IL-1β mRNA. Also consistent with the protein levels, DMSO did not increase the mRNA levels for TNF-α or IL-6. The response pattern of IL-1α mRNA was similar to that observed for TNF-α and IL-6. These results closely parallel the protein data, indicating that the mechanism of DMSO augmentation of IL-1β production occurs through augmentation of the mRNA coding for IL-1β.

FIGURE 6.

Effect of DMSO on LPS-stimulated mRNA cytokine expression. PBMC were preincubated with different concentrations of DMSO (0.5–1.0%) immediately before LPS stimulation. LPS (50 ng/ml) was added, and the incubation continued for 3 h. The levels of cytokine mRNA were analyzed by RPA. A, Representative autoradiogram: (1) 32P-labeled probes alone; (2) No LPS control; (3) 1.0% DMSO alone; (4) LPS-stimulated; (5) LPS plus 0.5% DMSO; (6) LPS plus 1.0% DMSO. B, Results are mean ± SEM (n = 3) of densitometric scanning of blots probed for different cytokines and corrected for the intensity of the L32 bands. ∗, p < 0.05 compared with LPS alone.

FIGURE 6.

Effect of DMSO on LPS-stimulated mRNA cytokine expression. PBMC were preincubated with different concentrations of DMSO (0.5–1.0%) immediately before LPS stimulation. LPS (50 ng/ml) was added, and the incubation continued for 3 h. The levels of cytokine mRNA were analyzed by RPA. A, Representative autoradiogram: (1) 32P-labeled probes alone; (2) No LPS control; (3) 1.0% DMSO alone; (4) LPS-stimulated; (5) LPS plus 0.5% DMSO; (6) LPS plus 1.0% DMSO. B, Results are mean ± SEM (n = 3) of densitometric scanning of blots probed for different cytokines and corrected for the intensity of the L32 bands. ∗, p < 0.05 compared with LPS alone.

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To determine whether the augmentation by DMSO on LPS-induced IL-1β mRNA required protein synthesis, we pretreated PBMC with the protein synthesis inhibitor cycloheximide (10 μg/ml) for 30 min followed by LPS alone or DMSO plus LPS treatment for 2 h. RNA was extracted for IL-1β mRNA analysis by RPA. As previously noted and reported (36), a small amount of IL-1β was detectable in untreated PBMC. As shown in Fig. 7, pretreatment with cycloheximide did not affect the LPS induction of IL-1β mRNA. DMSO augmentation of LPS-induced IL-1β mRNA was not altered by cycloheximide pretreatment, indicating that the DMSO enhanced IL-1β mRNA did not require de novo protein synthesis.

FIGURE 7.

Cycloheximide (CHX) does not alter the DMSO augmentation of LPS-stimulated IL-1β mRNA. PBMC were preincubated with 10 μg/ml cycloheximide for 30 min before 3 h of stimulation with LPS (50 ng/ml) or LPS+DMSO (1.0%). The levels of cytokine mRNA were analyzed by RPA. The results are the average of four independent experiments.

FIGURE 7.

Cycloheximide (CHX) does not alter the DMSO augmentation of LPS-stimulated IL-1β mRNA. PBMC were preincubated with 10 μg/ml cycloheximide for 30 min before 3 h of stimulation with LPS (50 ng/ml) or LPS+DMSO (1.0%). The levels of cytokine mRNA were analyzed by RPA. The results are the average of four independent experiments.

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Because the data indicated that DMSO increased steady state IL-1β mRNA, we sought to determine whether this increase in mRNA was due to a reduction in the degradation of the IL-1β mRNA. Cells were stimulated with LPS in the presence or absence of DMSO for 3 h and the transcriptional inhibitor, actinomycin D, was then added to the cultures. As expected, DMSO increased the levels of IL-1β mRNA (time 0 measurement). Samples were collected at different time points to determine the rates of IL-1β mRNA, which would occur after new transcription was halted. As shown in Fig. 8, LPS-induced IL-1β mRNA decayed quickly after the addition of actinomycin D. The mean half-life of IL-1β mRNA in LPS-treated and LPS plus DMSO-treated PBMC was 4.62 ± 0.25 and 4.50 ± 0.21 (n = 4, p > 0.05), respectively, indicating that DMSO did not alter the stability of IL-1β mRNA.

FIGURE 8.

Effect of DMSO on the stability of IL-1β mRNA. PBMC were stimulated with LPS or LPS+DMSO for 3 h before addition of actinomycin D. The time that actinomycin D was added was set as 0 h. The figure represents four experiments and displays the linear regression of IL-1β mRNA decay. There was no difference in the slopes of the curves or the calculated mRNA half life comparing LPS vs LPS+DMSO.

FIGURE 8.

Effect of DMSO on the stability of IL-1β mRNA. PBMC were stimulated with LPS or LPS+DMSO for 3 h before addition of actinomycin D. The time that actinomycin D was added was set as 0 h. The figure represents four experiments and displays the linear regression of IL-1β mRNA decay. There was no difference in the slopes of the curves or the calculated mRNA half life comparing LPS vs LPS+DMSO.

Close modal

Because DMSO increased steady state IL-1β mRNA and did not decrease degradation, we designed experiments to test whether DMSO augmented the activity of the IL-1β promoter. To identify whether DMSO affected LPS-induced promoter activity of the IL-1β gene, we constructed a reporter vector, pGL-1-IL-1β, using the pGL-3 basic vector and the whole known 5′-flanking region of the human IL-1β gene. For this set of experiments the murine monocyte cell line RAW 264.7 was used to allow optimal cell transfection with the reporter vectors. This cell line was also chosen for our experiments because the 15-kb human pro-IL-1β gene, corresponding to the entire IL-1β structural gene and flanking sequences, is regulated in a manner that is indistinguishable from the endogenous murine gene when stably introduced into RAW 264.7 cells (31, 37). RAW cells were cotransfected with pGL-3-IL-1β and with the pRL-TK vector as an internal control. Using previously reported data as a guideline (31, 37), the cells were stimulated with 10 μg/ml LPS in the presence or absence of DMSO for 12 h. As shown in Fig. 9, LPS induced a significant increase of the transcriptional response of human IL-1β promoter in murine RAW 264.7 cells. DMSO substantially augmented the LPS-induced transcriptional response as indicated by increased reporter activity. These data demonstrate that DMSO elevates IL-1β gene expression by acting directly at the level of gene transcription.

FIGURE 9.

DMSO increases human IL-1β promoter activity in response to LPS. RAW264.7 cells were cotransfected with both the pGL-1-IL-1β and the control vector pRL-TK. Firefly and Renilla luciferase activities in cell extracts were detected using the dual luciferase assay system. The activities of reporter vectors were expressed as the ratio of firefly and Renilla activity. LPS alone stimulated promoter activity that was further augmented by DMSO. ∗, p < 0.01 compared with control; #, p < 0.05 compared with the LPS-stimulated group.

FIGURE 9.

DMSO increases human IL-1β promoter activity in response to LPS. RAW264.7 cells were cotransfected with both the pGL-1-IL-1β and the control vector pRL-TK. Firefly and Renilla luciferase activities in cell extracts were detected using the dual luciferase assay system. The activities of reporter vectors were expressed as the ratio of firefly and Renilla activity. LPS alone stimulated promoter activity that was further augmented by DMSO. ∗, p < 0.01 compared with control; #, p < 0.05 compared with the LPS-stimulated group.

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Our previous studies and the present results demonstrate that DMSO increases LPS-induced IL-1β production in human blood (24, 25) and separated PBMC. To extend these findings to an in vivo setting, we tested the ability of DMSO to augment LPS-induced plasma levels of IL-1β. As shown in Fig. 10, a 10 μg/mouse challenge of LPS induced plasma IL-1β. Using a DMSO protocol demonstrated to modulate the inflammatory response (38, 39), mice were injected with DMSO (1.5 ml/kg) 10 min before LPS challenge, and plasma was obtained at 2 h post LPS. In this in vivo setting, DMSO significantly increased plasma levels of IL-1β. These data demonstrate that DMSO has the capacity to augment IL-1β production both in vitro and in vivo.

FIGURE 10.

DMSO augments LPS-stimulated plasma IL-1β in vivo. DMSO (1.5 ml/kg) was administered i.p. 10 min before LPS challenge (10 μg/mouse). Blood was collected 2 h after LPS treatment. LPS induced plasma levels of IL-1β. DMSO augmented the LPS-induced plasma levels of IL-1β. Values are mean ± SEM and n = 6 for LPS and LPS+DMSO. ∗, p < 0.05 compared with LPS-treated group.

FIGURE 10.

DMSO augments LPS-stimulated plasma IL-1β in vivo. DMSO (1.5 ml/kg) was administered i.p. 10 min before LPS challenge (10 μg/mouse). Blood was collected 2 h after LPS treatment. LPS induced plasma levels of IL-1β. DMSO augmented the LPS-induced plasma levels of IL-1β. Values are mean ± SEM and n = 6 for LPS and LPS+DMSO. ∗, p < 0.05 compared with LPS-treated group.

Close modal

It was previously reported that DMSO increases IL-1β secretion in the LPS-stimulated whole blood model (24, 25), although it inhibited IL-8 production. In the present study, we further demonstrated that DMSO augmented LPS-induced IL-1β production by human PBMC. An important aim of this study was to determine the molecular levels at which DMSO increased LPS-induced IL-1β production.

Substantial evidence exists supporting the concept that antioxidants, including synthetic and natural products, suppress cytokine production by monocytes/macrophages (20, 32). These previous papers indicate that ROIs play an important role in LPS-induced cytokine production. Koga et al. (34) reported that ROIs, such as superoxide anions, are involved in the pathways that stimulate IL-1β production. Previous work indicates that hydrogen peroxide primes the promonocytic U937 cell line to produce IL-1β (40). Hsu and Wen (41) showed that H2O2 plays an important role in LPS-induced IL-1β production. Many of the physiologic and pharmacologic properties of DMSO are related to its radical scavenger activity, such as radioprotection, ischemic protection, and anti-inflammatory effects (reviewed in Ref.42). As an antioxidant and hydroxyl radical scavenger, DMSO has been well described as inhibiting cytokine and chemokine production. However, our present study clearly demonstrated that DMSO enhanced LPS-induced IL-1β production, and this effect was due to an increase in steady state mRNA levels. The augmented mRNA was the direct result of increased IL-1β promoter activity.

Similar to the publications about other cytokines, conflicting reports exist about the effect of antioxidants and the regulation of IL-1β production. Parmentier et al. (43) reported that the antioxidant compound, NAC enhances LPS-induced IL-1β production from the human monocytic cell line THP-1 at the transcriptional level. They also demonstrated that the NAC-enhanced LPS-induced IL-1β release was dependent on the synthesis of new proteins, suggestive of posttranscriptional regulation by NAC. Mathy-Hartert et al. (44) observed that NAC increased LPS-induced IL-1β and inducible NO synthase in human chondrocytes. Consistent with these reports, our results indicate that NAC and DMTU augmented LPS-induced IL-1β production in this study. In contrast, Hsu and Wen (41) published that NAC decreased LPS-induced IL-1β production in the murine macrophage cell line J774A.1 by inhibiting ROI and modulating the ICE. A possible explanation for these discordant results may be that the effect of NAC is critically dependent upon concentration, cell type, and stimulus. Brennan and O’Neill (45) reported that oxidants, including hydrogen peroxide and the pro-oxidant diamide, and antioxidants, including NAC and pyrolidine dithiocarbamate, have different effects on three cell lines, demonstrating the effects of oxidants and antioxidants are restricted to certain cell types. The DMSO augmentation of LPS-induced IL-1β was first found in human whole blood model (25), which closely reproduces in vivo settings. In this study, primary cultures of human PBMC were used to reproduce the phenomena observed in whole blood model and examine more mechanistic studies of the cellular regulations. Our in vivo data extend the cell culture data and confirm that the antioxidant will increase IL-1β production in a whole animal model of acute inflammation.

In the process of IL-1 maturation, a precursor molecule referred to as pro-IL-1 is produced in the cytosol of macrophages or monocytes. The pro-IL-1 is a 31–34-kDa nonactive form of cytokine, enzymatically cleaved into a 17-kDa mature functional form by the ICE, now known as caspase 1 (3, 5, 6, 46). After the caspase cleavage, active IL-1β is released and exhibits its diverse biologic functions. Our data showed that DMSO increased the levels of mature IL-1β in the supernatant as well as increasing the levels of mRNA. However, there was minimal alteration in the amount of pro-IL-1β, which agrees with the studies that pro-IL-1β, is rapidly processed to be secreted outside the cell. The present study also shows that cycloheximide does not alter DMSO-enhanced IL-1β mRNA, suggesting new protein synthesis is not required for this phenomenon. The data with actinomycin D treatment shows that there is no difference in the degradation rate of IL-1β mRNA in the presence or absence of DMSO, demonstrating that DMSO does not increase the stability of IL-1β mRNA in PBMC. Because there is an increase in the total amount of IL-1β mRNA without a decrease in the degradation, this suggests that the oxidants are exerting a direct effect at the promoter level. To test the hypothesis that the effect of DMSO acts at the level of transcription, we used a reporter vector containing the whole known 5′-flanking region of IL-1β gene. This provides clear evidence showing that DMSO increased the promoter activity of IL-1β. We demonstrate in this model that treatment with DMSO significantly enhanced the LPS-induced reporter activity of the IL-1β promoter.

A number of reports have described the involvement of different nuclear transcription factors in IL-1β gene expression (30, 31, 35, 47). In this study, activation of two members of the NF-κB protein family was analyzed in the nuclear extracts. Consistent with reported data (35), p65 (RelA) and p50 (NF-κB1) were activated in PBMC in response to LPS. However, DMSO did not alter LPS-induced activation of NF-κB, indicating that the augmentation of DMSO on IL-1α gene expression is not through NF-κB at the transcription level. In vivo studies show that DMSO inhibits hepatic NF-κB activity in septic and hepatic fibrosis rat models (48, 49, 50, 51). However, in the A549 cell line, DMSO did not inhibit respiratory syncytial virus induced NF-κB binding activity (52). Vlahopoulos et al. (53) reported that 2% DMSO had no effect on inducible p65 (RelA) binding in human monocyte-like U937 cell line. It was also reported that some oxidants and antioxidants have different effects on NF-κB activity in different cells (43). The results reported in our studies are in agreement with these previous reports demonstrating that DMSO does not alter the levels of NF-κB.

IL-1β is often considered to be an important proinflammatory cytokine. The expression of IL-1β is closely controlled in healthy tissue and it has been postulated to represent an important central cytokine in inflammatory diseases (54). Despite numerous reports regarding the importance of IL-1β in the cytokine network, much remains to be learned about the mechanisms governing its expression. Our results demonstrate that production of IL-1β by LPS-stimulated human PBMC is enhanced by DMSO at both the transcriptional and posttranslational levels. An understanding of the basic mechanisms involved in the up-regulation of IL-1β following DMSO treatment may lead to experimental approaches for regulating IL-1β expression.

The authors have no financial conflict of interest.

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

1

This work was partially supported by National Institutes of Health Grant GM50901.

3

Abbreviations used in this paper: ICE, IL-1 converting enzyme; RPA, RNase protection assay; ROI, reactive oxygen intermediate; NAC, N-acetyl-l-cysteine; DMTU, dimethylthiourea.

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