Objective: The release of inflammatory cytokines from antigen-stimulated cells of the immune system is inhibited by resin monomers such as 2-hydroxyethyl methacrylate (HEMA). Although the formation of oxidative stress in cells exposed to HEMA is firmly established, the mechanism behind the inhibited cytokine secretion is only partly known. The present investigation presents evidence regarding the role of HEMA-induced oxidative stress in the secretion of the pro-inflammatory cytokine TNFα from cells exposed to the antigens LTA (lipoteichoic acid) or LPS (lipopolysaccharide) of cariogenic microorganisms using BSO (L-buthionine sulfoximine) or NAC (N-acetyl cysteine) to inhibit or stabilize the amounts of the antioxidant glutathione.
Method: RAW264.7 mouse macrophages were treated with LTA, LPS or HEMA in the presence of BSO or NAC for 1h or 24h. Secretion of TNFα from cell cultures was analyzed by ELISA, and the formation of reactive oxygen (ROS) or nitrogen species (RNS) was determined by flow cytometry. Protein expression was detected by Western blotting.
Results: The release of TNFα in both LTA- and LPS-exposed cells was decreased by HEMA, and this concentration-dependent inhibitory effect was amplified by BSO or NAC. LTA- and LPS-stimulated expression of the redox-sensitive transcription factor NF-αB (p65) in cell nuclei decreased in the presence of HEMA because the translocation of p65 from the cytosol was prevented by oxidative stress specifically increased by the monomer.
Conclusions: A disturbance of the cellular redox balance, particularly induced by HEMA, is a crucial factor in the inhibition of LTA- and LPS-stimulated signalling pathways leading to TNFα secretion.
Dental caries is an infectious disease generated by a plethora of Gram-positive and Gram-negative oral microorganisms in diversified biofilms on tooth surfaces. Invasion of dentinal hard tissue by pathogenic bacteria or their compounds LPS (lipopolysaccharide) or LTA (lipoteichoic acid) across dentinal tubules triggers the activation of cells of the innate immune system in the dental pulp complex [ , ]. While LPS is a glycolipid and a pathogenic molecule of Gram-negative cariogenic bacteria, LTA is a glycolipid connected to polyglycerolphosphate in the cell wall of Gram-positive microbes. As potent inflammatory PAMPs (pathogen-associated molecular patterns), LPS and LTA bind to Toll-like receptors (TLR) on the surface of cells of the dental pulp, such as odontoblasts at the dentin-pulp interface, and tissue resident immune cells such as fibroblasts, dendritic cells and macrophages. Binding of LPS to TLR4 and LTA to TLR2/TLR6 receptors triggers signaling cascades through MyD88-dependent pathways, finally activating the redox-sensitive transcription factor NF-κB (nuclear factor-κB) or MAPKs, and resulting in the production and release of pro- or anti-inflammatory cytokines [ ]. Although the immediate cell response to LPS or LTA exposure varies depending on the cell type and distinct signaling components, both molecules stimulate the release of cytokines such as TNFα in macrophages [ , ]. The release of cytokines as a result of signaling through NF-κB is positively or negatively regulated by reactive oxygen (ROS) or nitrogen species (RNS) such as hydrogen peroxide or nitric oxide (NO), and a related overabundance of reactive molecules. The formation of ROS and RNS under proinflammatory conditions, in turn, is mostly a result of the enhanced NFκB-regulated expression of Nox2 and iNOS enzymes [ , ]. Therefore, it seems as if LPS- or LTA-stimulated activation of NFκ-B and the formation of inflammatory cytokines was the cause and effect of oxidative stress [ , ]. Downregulation of LPS- or LTA-stimulated inflammatory processes, on the other hand, is related to the NFκB- and stress-induced expression of the antioxidant protein HO-1 (hemeoxygenase 1), which protects cells from ROS and RNS [ , , ]. In addition to NFκB, the expression of HO-1 is directly controlled by the transcription factor Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2), which is a master regulator of cellular redox homeostasis. Consequently, HO-1 is considered to be the mediator of an NFκB-Nrf2 crosstalk [ , ].
As a basic mechanism, dental resin monomers such as TEGDMA or HEMA induce intracellular oxidative stress by the enhanced formation of ROS and RNS as repeatedly shown [ ]. It is also firmly established that HO-1, catalase and related enzymes are activated under the control of Nrf2 as an adaptive response in order to reestablish cellular redox homeostasis and vital cell functions [ ]. Monomer-induced oxidative stress beyond the capacities of the Nrf2-coordinated antioxidant network results in cytotoxic effects via apoptosis, at least in part as a consequence of oxidative DNA damage [ , ]. The crucial importance of Nrf2 activity for maintaining redox homeostasis and cell viability was impressively demonstrated through its stimulation by the artificial compound tBHQ ( tert -butylhydroquinone) [ ]. It has also been suggested that HEMA might activate Nrf2 via its electrophilic properties [ ]. Still, using tBHQ as an inducer of Nrf2 activation also provided evidence that monomer-induced oxidative stress was also associated with the inhibition of the release of pro- and anti-inflammatory cytokines from LPS-stimulated immunecompetent macrophages [ ].
It has repeatedly been shown that the release of TNFα, IL-6 and IL-10 from LPS-stimulated cells was inhibited in the presence of increasing concentrations of HEMA [ , ]. The mechanism behind this is the inhibition of the LPS-stimulated translocation of NF-κB from the cytosol to the cell nucleus in the presence of the monomer [ ]. Analysis of the formation of reactive oxygen and nitrogen species suggested that molecules such as H 2 O 2 and NO enhanced in HEMA-treated cells prevented the activation of NF-κB and subsequent cytokine production [ ]. Therefore, in the present investigation we analyzed in more detail the role of oxidative stress in the inhibition of the release of cytokines relevant for vital immune responses to invading cariogenic pathogens by resin monomers. To this end, we modified the capacity of cells to control the intracellular redox balance through the modification of the concentration of glutathione (GSH) as a major non-enzymatic antioxidant. We have previously shown that oxidative stress increased when GSH synthesis was inhibited by buthionine sulfoximine (BSO), but decreased when GSH levels were maintained at high levels in the presence of N -acetyl cysteine (NAC), a substrate in GSH synthesis [ ]. Here, the effects of LPS and LTA on cytokine secretion, formation of ROS and RNS, as well as expression levels of related proteins including the redox-sensitive transcription factors NF-κB and Nrf2 were investigated. These parameters have not been analyzed thus far in cells treated with LTA. The secretion of the cytokine TNFα from RAW264.7 mouse macrophages was used as a reliable and approved model of immune cell function as previously shown [ ]
Materials and methods
Reagents and antibodies
2-Hydroxyethyl methacrylate (HEMA; CAS-No. 868-779) was obtained from Merck (Darmstadt, Germany). RPMI 1640 medium ( l -glutamine, 2.0 g/l NaHCO 3 ) was purchased from PAN Biotech (Aidenbach, Germany). Fetal bovine serum (FBS) was obtained from Life Technologies, Gibco BRL (Eggenstein, Germany). Lipopolysaccharide (LPS; E. coli , serotype 055:B5), dihydroethidium (DHE), lipoteichoic acid from Staphylococcus aureus (LTA; CAS-No. 56411-57-5) dihydrorhodamine 123 (DHR123), l -buthionine sulfoximine (BSO; CAS-No. 83730-53-4), N -acetyl cysteine (NAC; CAS-No. 616-91-1), 3-(4,5 dimethyiazol-2-1)-2-5-diphenyl tetrazolium bromide (MTT), crystal violet (C0775), and a bicinchoninic acid protein assay kit came from Sigma (Taufkirchen, Germany). 2′7′-dichlorodihydrofluorescin diacetate (DCFH 2 -DA; CAS-No. 4091-99-0) was obtained from MoBiTec (Göttingen, Germany). Amido black 10B was obtained from Merck (Darmstadt, Germany). Anti-catalase (H-300, sc-50508), anti-heme oxygenase 1 (HO-1, M-19, sc-1797), anti-Nrf2 (H-300, sc-13032), anti-p47-phox (sc-17845) monoclonal antibodies, and a protease inhibitor cocktail were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-NF-κB p65 (no. 6956), anti-phospho-NF-κB p65 (no. 3033), anti-iNOS (D6B6S, no. 3120), plus anti-rabbit IgG HRP-linked antibodies (no. 7074) came from cell signaling (NEB Frankfurt, Germany). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (clone 6C5) was obtained from Millipore (Schwalbach, Germany).
Goat anti-mouse IgG (H + L)-HRP conjugate was purchased from Bio-Rad Laboratories (Munich, Germany), and Amersham hyperfilm ECL came from GE Healthcare (Munich, Germany). Cell Meter™ fluorimetric intracellular peroxynitrite (no. 16317) and Cell Meter™ fluorimetric intracellular nitric oxide (NO) (no. 16356) assay kits were purchased from AAT Bioquest (Sunnyvale, CA, USA). The protease inhibitor cocktail (complete mini) was obtained from Roche Diagnostics (Mannheim, Germany), and a tumor necrosis factor-α (TNF-α) ELISA kit was obtained from BD Biosciences (San Diego, CA, USA).
The murine RAW264.7 mouse macrophages cell line (ATCC TIB71) was maintained in RPMI 1640 medium supplemented with l -glutamine, sodium-pyruvate, 2.0 g/l NaHCO 3 and 10% fetal bovine serum (FBS) following our standard culture techniques as previously described [ ].
Determination of TNFα
Cells (5 × 10 4 cells/well) from routine culture were incubated in RPMI 1640 medium in 6-well plates at 37 °C for 48 h. For the analysis of concentrations of LPS or LTA effective in the release of tumor necrosis factor-α (TNFα) from RAW264.7 mouse macrophages, cell cultures were exposed to LTA or LPS in the range of 0–50 μg/ml in 2 ml culture media. Next, cell cultures were treated with 50 μM BSO (L-buthionine sulfoximine) or 10 mM NAC ( N -acetyl cysteine) to analyze for the role of glutathione in the release of TNFα from HEMA-exposed cells. To this end, cell cultures were initially preincubated with 50 μM BSO for 18 h or with 10 mM NAC ( N -acetyl cysteine) for 1 h. Then, preincubation media were removed and cells were exposed to HEMA (0–1–4–8 mM), both in the presence or absence of LTA (10 μg/ml) or LPS (0.1 μg/ml), or 50 μM BSO or 10 mM NAC in 2 ml culture media as described [ ]. Cell culture supernatants were collected after 1 h or 24 h exposure periods and analyzed for the formation of tumor necrosis factor-α (TNF-α). In addition, amounts of proteins present in adherent cells were analyzed using an Amido Black assay as described below (Section 2.4 ). BSO and NAC concentrations appropriate under the current experimental conditions were established in a previous investigation [ ]. Concentrations of LTA and LPS were chosen because of their effectiveness in the release of TNF-α observed in range finding experiments shown in Fig. 1 .
Amounts of TNF-α (pg/ml) were determined using a standard ELISA kit (BD Pharmingen). Absorbance was read at 450 nm in a spectrophotometer (Infinite F200, TECAN, Mainz, Germany), and optical density values were collected (Magellan software; version 6.2). Levels of TNF-α in untreated cell cultures below the detection limit were set to the lowest detectable levels (15.6 pg/ml) in standard curves. Absolute amounts of TNFα were summarized from duplicate values in 4–5 independent experiments as specified in the figure legend.
Determination of protein concentration
Adherent cells in 6-well plates employed for the analysis of TNF-α release in cultures treated with HEMA and LTA/LPS were washed with phosphate-buffered saline (PBS/5 mM EDTA), and then lysed in 500 μl RIPA lysis buffer/well for 10 min in the cold. The cell lysate was then transferred to an assay tube. Each well was washed with 400 μl RIPA lysis buffer and the buffer solution was then combined with the cell lysate. Protein concentration in each sample was then determined with Amido Black as described earlier [ ]. 500 μl of reagent (13 μg/ml Amido Black/ml, dissolved in 10% acetic acid, 90% methanol) was added to 80 μl of protein sample and incubated for 5 min at room temperature. Then, the solution was centrifuged at 16,000× g for 5 min, the supernatant discarded, the pellet washed in 500 μl of 10% acetic acid, 90% methanol, and again centrifuged at 16,000× g for 5 min. The washing procedure was repeated twice, and finally the pellet was dissolved in 400 μl of 0.1 N NaOH. An aliquot of 180 μl of each sample was individually transferred to wells in a 96-well plate, and the absorbance was determined at 615 nm in a spectrophotometer (Tecan). Absolute amounts of proteins were calculated from standard curves established with bovine serum albumin (0–30 μg).
Detection of reactive oxygen species (ROS)
Cell cultures (5 × 10 4 /well) were maintained in routine culture medium in 6-well plates for 48 h, and then preincubated with 50 μM BSO for 18 h, or with 10 mM NAC ( N -acetyl cysteine) for 1 h. Subsequently, cell cultures were treated with HEMA (0–1–4–8 mM), both in the presence or absence of LTA (10 μg/ml) or LPS (0.1 μg/ml), or 50 μM BSO or 10 mM NAC in 2 ml culture media [ ]. The exposure of cells was stopped by discarding the exposure media, and the formation of ROS was analyzed by flow cytometry after staining the cells with the fluorescent dyes 2′7′-dichlorodihydrofluorescin diacetate (DCFH 2 -DA), dihydroethidium (DHE) or dihydrorhodamine 123 (DHR123). It has been previously reported that DCFH 2 -DA indicated the formation of general oxidative stress, while DHE or DHR123 detected the formation of superoxide anions or hydrogen peroxide production as discussed in our recent investigation [ ]. Favorable properties of these dyes for the investigation of monomer-induced oxidative stress have been outlined in detail in our previous publication [ ]. Cells were incubated with 10 μM DCFH 2 -DA, 5 μM DHE or 5 μM DHR123 in culture medium 30 min prior to harvesting in phosphate-buffered saline (PBS)/5 mM EDTA. Then, the cells were collected by centrifugation and the cell pellet was resuspended in 200 μl CMF–PBS. DCF and DHR123 fluorescence was measured in the FL-1 channel (488/515–545) using a BD FACSCanto (Becton Dickinson) flow cytometer, and DHE fluorescence intensities were detected in Fl-2 (488/546–606). Mean fluorescence intensities (MFI) were established using histogram statistics and FACSDiva™ 5.0.2 (Becton Dickinson) software. Individual fluorescence intensities measured in treated cell cultures were related to fluorescence quantified in untreated control cultures (= 1.0) as described [ ].
Analysis of nitric oxide and peroxynitrite formation
The cells (5 × 10 4 /well) were grown in routine culture medium in 6-well plates for 48 h, and then preincubated with 50 μM BSO or 10 mM NAC ( N -acetyl cysteine), exposed to HEMA (0–1–4–8 mM) in the presence or absence of LPS (0.1 μg/ml) or LTA (10 μg/ml), or 50 μM BSO or 10 mM NAC as specifically described in Section 2.5 . Exposure of cells was stopped by discarding the exposure media, and cells were then harvested in phosphate-buffered saline (PBS)/5 mM EDTA, collected by centrifugation and resuspended in 200 μl CMF–PBS. The intracellular formation of nitric oxide (NO ) and peroxynitrite (ONOO) was determined using Cell Meter™ fluorimetric assay kits (AAT Bioquest). Peroxynitrite (ONOO ) or nitric oxide (NO) was examined by flow cytometry (FACSCanto) in channels FL-1 (488/519 nm, ONOO ) or FL-5 (633/660, NO) following the manufacturer’s instructions and as described elsewhere [ ]. Mean fluorescence intensities were determined, and individual fluorescence intensities normalized as described in Section 2.5 .
Analysis of protein expression by immunoblotting
Cells (1.5 × 10 6 cells) were grown in routine culture medium on plates (150 mm diameter) at 37 °C for 48 h. Next, cell cultures were preincubated with 50 μM BSO or 10 mM NAC ( N -acetyl cysteine), exposed to HEMA (0–1–4–8 mM) in the presence or absence of LPS (0.1 μg/ml) or LTA (10 μg/ml), or 50 μM BSO or 10 mM NAC in culture medium. Exposure of cells was stopped by discarding the exposure media. Floating and adherent cells were harvested in ice-cold PBS and combined after centrifugation. Nuclear and cytosolic cell fractions were prepared using different lysis buffers and various centrifugation procedures as described in our previous paper [ ]. Briefly, cell pellets were resuspended in 1 ml ice-cold PBS, mixed with 0.5 ml buffer A (10 mM Tris HCl, 60 mM KCl, 1 mM Na 2 EDTA, 1 mM DTT, pH 7.4), and incubated on ice. The cell suspension was centrifuged again, and the cell pellet was solubilized in buffer B (buffer A plus 0.4% NP 40, 5 mM NaF, 1 mM NaVO 4 ) in the cold. After centrifugation (14,000× g ) the supernatant was collected as a cytoplasmic cell fraction. The cell pellet containing cell nuclei in the pellet was washed in buffer A, resuspended in buffer C (20 mM Tris-Cl pH 8.0, 400 mM NaCl, 1.5 mM MgCl 2 , 1.5 mM Na 2 EDTA, 25% glycerol, 1 mM DTT, 5 mM NaF, 1 mM NaVO 4 ), and centrifugated (14,000× g ) again in the cold. The resulting supernatant was collected as a fraction of nuclear proteins. Protein amounts in the cytosolic and nuclear cell fractions were measured using a BCA protein assay.
Protein expression was identified by routine immunoblotting as previously described [ ]. Proteins (10–15 μg per lane) were first separated by SDS-PAGE and then transferred to a nitrocellulose membrane. After washing in TBS (25 mM Tris-Cl, 150 mM NaCl, pH 7.4) and blocking with 5% nonfat milk in TBST (TBS plus 0.1% Tween 20, pH 7.4) at room temperature, the membrane was incubated with primary antibodies to detect protein expression as specified in the legends of Figs. 4 and 7 . Primary antibodies bound to membranes were visualized by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL) using a blot scanner and Image Studio software (LI-COR Biotechnology, Bad Homburg, Germany). Membranes were stripped with CHEMICON re-blot stripping solution (Millipore, Schwalbach, Germany) for reprobing.
Individual values from repeated independent experiments as specified in the figure legends were summarized as medians (25–75% quartiles). The Mann–Whitney U -test (SPSS Statistics 23, IBM, Armonk, NY, USA) with an alpha level of 0.05 was used to calculate statistically significant differences between median values.
Effectiveness of LPS and LTA in the stimulation of TNFα secretion
The effectiveness of LTA (lipoteichoic acid) and LPS (lipopolysaccharide) in the triggering of innate immune responses was initially compared by the stimulation of TNFα secretion in RAW264.7 mouse macrophages through a wide range of LTA or LPS concentrations. While the amounts of TNFα detected in untreated cell cultures were close to the detection limit, both LTA and LPS were differentially effective in the stimulation of TNFα release depending on concentrations and the exposure period. LTA induced a concentration-dependent increase in TNFα release after 1 h or 24 h exposure periods. LTA concentrations in the range of 0.1–50 μg/ml significantly increased TNFα secretion more than 30-fold to about 200–300 pg/ml after a 1 h exposure, and TNFα release further increased about 3-fold when cells were exposed to high LTA concentration for a long exposure period (24 h) ( Fig. 1 A, B). LPS concentrations from 0.1 to 50 μg/ml significantly, and almost equally, increased TNFα secretion to about 500–600 pg/ml after a 1 h exposure, and TNFα release further increased about 5–10-fold after a long exposure period (24 h) ( Fig. 1 C, D).
Despite the clear influence on TNFα secretion, no influence on cell survival was detected with all LTA or LPS concentrations after a 1 h exposure. Cell survival was moderately reduced to about 70–75% by LPS (0.1–50 μg/ml), while no decrease in cell survival was observed with LTA (0.1–50 μg/ml) after a 24 h exposure ( Fig. 1 E, F). Concentrations of 10 μg/ml LTA and 0.1 μg/ml LPS were then selected to further investigate the influence of the monomer HEMA on LPS- or LTA-stimulated TNFα secretion.
LTA-stimulated secretion of TNFα and oxidative stress conditions
LTA-stimulated secretion of TNFα
No increase in the amounts of TNFα set to the detection limit in untreated cell cultures was detected after the exposure of cells to increasing HEMA concentrations for 1 h or 24 h. On the contrary, the considerable increase in the secretion of absolute amounts of 374 pg/ml or 465 pg/ml TNFα found in cultures stimulated with 10.0 μg/ml LTA for 1 h or 24 h was significantly inhibited in the presence of HEMA depending on its concentration ( Fig. 2 A, B). Both buthionine sulfoximine (BSO) and N -acetyl cysteine (NAC) were ineffective alone, but decreased LTA-stimulated TNFα secretion and affected the inhibitory effectiveness of HEMA as presented in detail below ( Fig. 2 A, B).
In order to normalize the absolute amounts of TNFα secreted under the various experimental conditions, we determined the amounts of protein present in exposed cell cultures ( Fig. 2 C, D). No significant influence of HEMA concentrations (0–8 mM) was found on the amounts of protein (60–84 μg) after a 1 h exposure period. While levels of proteins were only slightly reduced in cultures exposed to LTA in the presence of 4 or 8 mM HEMA, a decrease in protein content from about 84 μg (0 mM HEMA) to about 55–70 μg following a consistent pattern was observed in cultures treated with NAC, both in the presence or absence of LTA and HEMA ( Fig. 2 C). After a 24 h exposure the amount of protein increased in untreated cultures almost 4-fold to around 320 μg and was gradually decreased to 104 μg in cultures treated with 8 mM HEMA only ( Fig. 2 D). Noteworthy is that the protein levels increased to 134 μg in cultures co-treated with 8 mM HEMA and LTA compared to those exposed to 8 mM HEMA only. A continuous decrease in protein levels was observed with cultures treated with BSO in the presence or absence of LTA and HEMA down to about 50 μg in cells exposed to 8 mM HEMA. In comparison, 10 mM NAC alone reduced the amount of protein to about 190 μg, but unlike the effect of BSO, NAC increased protein levels when cell cultures were treated with a high concentration HEMA (8 mM) ( Fig. 2 D).
Related to these amounts of proteins, LTA-stimulated levels of TNFα decreased more than 3-fold from 9.2 pg LTA/μg protein after a 1 h exposure period to 2.8 pg LTA/μg protein after a 24 h exposure. Yet, secretion of TNFα was drastically and equally reduced with increasing HEMA concentrations. For instance, 2.8 pg LTA/μg protein detected in LTA-stimulated cultures were reduced to 1.1 and 0.4 or 0.3 pg LTA/μg protein in the presence of 1, 4 or 8 mM HEMA after a 24 h exposure. A similar pattern of TNFα secretion was detected after a 1 h exposure ( Fig. 2 E, F). The concentrations of BSO and NAC chosen here were highly effective in the inhibition the LTA-stimulated TNFα secretion after a 24 h exposure in particular. For instance, levels of TNFα in LTA-exposed cells decreased from 2.8 pg/μg protein to 1.5 (BSO) or 0.2 (NAC) pg/μg protein ( Fig. 2 F). Both substances seemed to intensify the inhibitory effectiveness of HEMA after long exposure (24 h), although no such effect was detectable with BSO after a 1 h exposure ( Fig. 2 E, F).
LTA-stimulated oxidative and nitrosative stress
The influence of BSO and NAC on the release of TNFα from LTA- and HEMA-stimulated cells after long exposure in particular suggested a crucial role of GSH and the production of ROS or RNS. Thus, the formation of oxidative stress, nitric oxide (NO) and peroxynitrite was further analyzed. Oxidative stress indicated by DCF fluorescence significantly increased in RAW264.7 mouse macrophages after exposure to increasing concentrations of HEMA for 1 h ( Fig. 3 A). A 1.3-fold increase compared to untreated cell cultures (= 1.0) was detected with 8 mM HEMA, and DCF fluorescence further increased more than 2−3-fold in the presence of BSO in cultures co-treated with HEMA ( Fig. 3 A). This effect of BSO was not detectibly influenced by 10 μg/ml LTA. LTA, however, slightly reduced DCF fluorescence alone and in the presence of HEMA. Furthermore, DCF fluorescence was also significantly reduced by NAC in cultures treated with HEMA, and this inhibitory effect was reproducibly intensified by LTA ( Fig. 3 A). Most relevant, a 5-fold increase in LTA-stimulated DCF-fluorescence after a 24 h exposure was continuously and significantly decreased by HEMA. This pattern of LTA-induced DCF fluorescence and inhibition in the presence of HEMA was drastically increased in the presence of BSO. LTA-stimulated DCF-fluorescence was considerably reduced in the presence of NAC, which then further intensified the inhibitory effect of HEMA ( Fig. 3 B)
Levels of nitric oxide (NO) were reduced to 0.9 in the presence of LTA compared to untreated cell cultures (= 1.0), and this effect was slightly but not significantly enhanced by 1 or 4 mM HEMA after a short exposure period (1 h) ( Fig. 3 C). While no influence of HEMA on the formation of NO · was detected in the presence or absence of BSO, NAC reduced the amounts of NO in control cultures and cells exposed to a low concentration of HEMA ( Fig. 3 C). After long exposure (24 h), LTA stimulated the formation of NO about 2.7-fold compared to untreated controls. This effect was reduced by 1 and 4 mM HEMA, but increased again with 8 mM HEMA to levels induced by LTA alone. HEMA itself significantly and continuously increased the formation of NO up to 3-fold compared to untreated cell cultures. The presence of BSO, which was ineffective alone and on LTA, even intensified the increase in NO formation in cell cultures treated with HEMA. In contrast, NAC reduced both LTA-stimulated and HEMA-induced production of NO ( Fig. 3 D). The levels of peroxynitrite (ONOO ) [ ], as a product of superoxide anion and nitric oxide, were slightly enhanced only in cultures treated with BSO for a short period of time ( Fig. 3 E, F). In contrast, ONOO formation was enhanced about 2-fold in cells stimulated with LTA compared to untreated cultures after a 24 h exposure. This LTA-induced effect, however, was moderately but significantly reduced with increasing concentrations of HEMA ( Fig. 3 F). The formation of peroxynitrite by LTA was independent of BSO, but BSO alone enhanced the ONOO levels about 1.3-fold, and it considerably (2-fold) intensified a slight increase in ONOO levels caused by HEMA. NAC reduced LTA-stimulated and HEMA-induced production of ONOO ( Fig. 3 F).
LTA-stimulated expression of proteins related to TNFα release and oxidative or nitrosative stress
Expression of proteins directly related to oxidative and nitrosative stress was detected in cell cultures exposed to LTA and HEMA for 24 h. The redoxsensitive transcription factor Nrf2 was expressed in the cytosol of untreated cell cultures, and Nrf2 expression was also detected in the nucleus in cells exposed to HEMA. Moreover, high levels of Nrf2, as detected in the nucleus of cells exposed to 10 μg/ml LTA possibly after translocation from the cytosol to the cell nucleus, were decreased in the presence of HEMA. Most relevant, expression of Nrf2 in the cell nucleus was drastically decreased in LTA-stimulated cultures co-exposed to the antioxidant NAC, while nuclear Nrf2 expression was moderately reduced in the presence of BSO ( Fig. 4 ). The expression of the transcription factor NF-κB, as indicated by subunit p65, was also stimulated in nuclei of cells treated with 10 μg/ml LTA, and these increased levels of NF-κB were reduced in the presence of HEMA. LTA-stimulated NF-κB expression also slightly decreased in the presence of NAC, as shown by the enhanced NF-κB levels in the cytosolic fraction compared to cultures treated with LTA alone. Although BSO alone strongly increased the expression of p65, the induction of nuclear NF-κB expression by LTA was even lower in cultures pretreated with BSO ( Fig. 4 ). Both HEMA and LTA induced expression of the stress protein HO-1, which was further enhanced in the presence of BSO. In contrast, co-treatment with NAC reduced HO-1 expression to low levels in cell cultures exposed to LTA or HEMA.
Expression of the inducible form of nitric oxide synthase (iNOS) was extremely low or absent under the current experimental conditions in cultures exposed to HEMA, NAC or BSO. Notably, a drastically increased expression of iNOS in LTA-stimulated cultures was effectively inhibited in the presence of HEMA. While LTA alone even lowered p47 expression, a remarkable increase in the basic expression of p47, as a regulatory subunit of NADPH oxidase, was detected in cells exposed to NAC. An increase in catalase expression in cell cultures exposed to HEMA was moderately reduced in the presence of LTA independent of the presence of NAC or BSO ( Fig. 4 ).