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Originally published online as doi:10.1189/jlb.1006599 on February 8, 2007

Published online before print February 8, 2007
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(Journal of Leukocyte Biology. 2007;81:1322-1332.)
© 2007 by Society for Leukocyte Biology

Hydrogen sulfide induces the synthesis of proinflammatory cytokines in human monocyte cell line U937 via the ERK-NF-{kappa}B pathway

Liang Zhi, Abel Damien Ang, Huili Zhang, Philip K. Moore and Madhav Bhatia1

Department of Pharmacology, National University of Singapore, Singapore

1 Correspondence: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Life Sciences, 28 Medical Drive, Singapore 117456. E-mail: mbhatia{at}nus.edu.sg


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hydrogen sulfide (H2S) is now considered an endogenous, gaseous mediator, which has been demonstrated to be involved in many inflammatory states. However, the mechanism of its proinflammatory function remains unknown. In the present study, we used IFN-{gamma}-primed human monocytic cell line U937 to investigate the effects of H2S in vitro on monocytes. We found that treatment with the H2S donor, sodium hydrosulfide, led to significant increases in the mRNA expression and protein production of TNF-{alpha}, IL-1ß, and IL-6 in U937 cells. H2S-triggered monocyte activation was confirmed further by the up-regulation of CD11b expression on the cell surface. We also observed that H2S could induce a rapid degradation of I{kappa}B{alpha} and subsequent activation of NF-{kappa}B p65, and this effect was attenuated by Bay 11-7082, a specific inhibitor of NF-{kappa}B. Furthermore, pretreatment of cells with Bay 11-7082 substantially inhibited the secretion of TNF-{alpha}, IL-1ß, and IL-6 induced by H2S. We also found that H2S stimulated the phosphorylation and activation of ERK1/2, but not of p38 MAPK and JNK, and pretreatment with PD98059, a selective MEK1 antagonist, could inhibit H2S-induced NF-{kappa}B activation markedly. Together, our findings suggest for the first time that H2S stimulates the activation of human monocytes with the generation of proinflammatory cytokines, and this response is, at least partially, through the ERK-NF-{kappa}B signaling pathway.

Key Words: H2S • inflammation • leukocyte • transcription factor • MAPK


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hydrogen sulfide (H2S) has been viewed traditionally as a toxic gas with the smell of rotten eggs found in the contaminated environmental atmosphere [1 ]. In recent years, however, there has been increased interest in endogenously generated H2S as a physiologically important molecule, which is suggested to be "the third endogenous gaseous signaling transmitter," alongside NO and carbon monoxide [2 3 4 ]. Now it is apparent that H2S is produced naturally in mammalian bodies, mainly by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine ß-synthase (CBS; EC 4.2.1.22) and cystathionine {gamma}-lyase (CSE; EC 4.4.1.1), using L-cysteine as the main substrate [5 , 6 ]. Physiological levels of H2S vary from 30 to 51 µM in rat serum [7 8 9 10 ], 11 to 35 µM in mouse serum [11 12 13 ], and 36 to 42 µM in human serum [14 , 15 ].

Recent studies have implicated that endogenous H2S plays an important role in many physiological and pathological processes, such as vasodilatation [9 , 16 17 18 19 20 21 ], cardioprotection [22 , 23 ], and neuromodulation [24 , 25 ]. Furthermore, H2S can reduce the contractility of isolated ileum [26 ] and attenuate the gastric mucosal injury induced by nonsteroidal anti-inflammatory drug (NSAID) [27 ].

Although the role of H2S as a vasodilator has been known for some time, the part played by H2S in inflammation has only begun to be addressed recently [4 ]. In the first report about the involvement of endogenous H2S in inflammation, we have shown that H2S fulfils an important, proinflammatory role in caerulein-induced acute pancreatitis and associated lung injury [11 ]. The severity of pancreatitis and subsequent lung injury are reduced markedly by treatment with DL-propargylglycine, an irreversible inhibitor of H2S-synthesizing enzyme CSE. In subsequent studies, we and other investigators have demonstrated a key role of H2S in carrageenan-induced hindpaw edema [28 ], LPS-induced endotoxemia [12 , 29 , 30 ], cecal ligation and puncture-induced sepsis [13 ], vanilloid receptor 1-mediated neurogenic inflammation [31 ], and chronic obstructive pulmonary disease [14 ].

Although the involvement of H2S in inflammation has become clear, little is known about the mechanism of its proinflammatory function. The effect of H2S on leukocyte activation should perhaps be considered. Leukocyte infiltration and proinflammatory mediator production are universal components of a wide range of diseases including inflammatory conditions. Amongst leukocytes, monocytes/macrophages play an important role in inflammation, angiogenesis, and atherosclerosis. Activated monocytes release preformed, proinflammatory mediators from granules and synthesize de novo cytokines and chemokines important in the amplification of inflammatory response. During this process, the inducible transcription factor NF-{kappa}B plays an essential role in controlling the transcription of the immune and inflammatory response genes [32 ]. In addition, as we know, MAPKs can act as the upstream activators of NF-{kappa}B [33 ].

Hence, the present study was designed to investigate whether H2S would provoke the activation of monocytes and whether NF-{kappa}B and MAPKs were involved in this process. The study was performed in vitro using the human monocyte model (IFN-{gamma}-primed human promonocytic cell line U937).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
U937 cells were kindly provided by Dr. Alirio J. Melendez (Department of Physiology, National University of Singapore). Cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT, USA), 2 mM glutamine, 10 units/ml penicillin, and 10 mg/ml streptomycin (Invitrogen). They were maintained in logarithmic growth by passage every 2–3 days. The viability of the cells (97–98%) was tested by the Trypan blue exclusion procedure. To differentiate U937 into monocyte, cells were primed with 200 ng/ml IFN-{gamma} (BioSource, Camarillo, CA, USA) for 18 h before the subsequent experiments.

Sodium hydrosulfide (NaHS), Bay 11-7082, and PD98059 treatment
Differentiated U937 cells were treated with the H2S donor, NaHS (Sigma Chemical Co., St. Louis, MO, USA), at the concentrations of 0.01, 0.1, or 1 mM, respectively, for the following stating times. This range of concentration for NaHS was chosen depending on the physiological level of H2S in the body. The solutions containing NaHS were prepared immediately before use from a 1 M NaHS stock solution. NaHS is widely used as a source of H2S. NaHS dissociates to Na+ and HS– in solution, and then HS– associates with H+ and produces H2S. The use of NaHS enables us to define the concentration of H2S in solution more accurately and reproducibly than bubbling H2S gas. As the active form of H2S (H2S, HS–, or S2–) has yet to be determined, the term "H2S" is used to reflect the sum of these three species. In some experiments, the NF-{kappa}B inhibitor Bay 11-7082 or MEK1 inhibitor PD98059 (Calbiochem, San Diego, CA, USA) was applied with preincubation for 1 h before NaHS treatment.

RT-PCR
Total RNA was extracted with the RNeasy® mini kit (Qiagen, Hilden, Germany) and reversely transcribed with the iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Then, cDNA was amplified by PCR in a thermal cycler (MyCyclerTM, Bio-Rad) using iQTM Supermix (Bio-Rad). The sequences of the primers were as follows: TNF-{alpha} sense, 5'-GGC TCC AGG CGG TGC TTG TTC-3', product size, 409 bp; TNF-{alpha} antisense, 5'-AGA CGG CGA TGC GGC TGA TG-3'; IL-1ß sense, 5'-CTT CAT CTT TGA AGA AGA ACC TAT CTT CTT-3', 332 bp; antisense, 5'-AAT TTT TGG GAT CTA CAC TCT CCA GCT GTA-3'; IL-6 sense, 5'-GAT GGA TGC TTC CAA TCT GGA T-3', 450 bp; antisense, 5'-AGT TCT CCA TAG AGA ACA ACA TA-3'; ß-actin sense, 5'-CCC AAG GCC AAC CGC GAG AAG AT-3', 219 bp; antisense, 5'-GTC CCG GCC AGC CAG GTC CAG-3'. PCR products were electrophoresed through 1.5% agarose gels containing 0.5 µg/ml ethidium bromide (Bio-Rad) and visualized by the UVP® bioimaging system (UVP, Upland, CA, USA). ß-Actin was used as an internal control to compare the amount of total mRNA of each sample used in the RT-PCR reaction. The intensity of bands was analyzed using LabWorksTM Image Analysis software (UVP).

ELISA
Cells were stimulated by the addition of NaHS. Following stimulation, the supernatants were collected and stored at –20°C until further use. TNF-{alpha}, IL-1ß, and IL-6 levels in the supernatants were evaluated by sandwich ELISA (DuoSetTM kit, R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Absorbance was measured at 450 nm by a microplate reader (Tecan Systems, San Jose, CA, USA). Results were shown as pg/ml of each cytokine.

Immunofluorescence
After treatment with NaHS, U937 cells were fixed in 3.7% formaldehyde and deposited on microscope slides using CytoFuge® 2 cytocentrifuge (StatSpin, Westwood, MA, USA). Then, the fixed cells were blocked for nonspecific binding with 1% BSA for 30 min at room temperature. Fluorescent labeling was performed by incubating the cells with a 1:50 dilution of mouse antihuman CD11b mAb (Serotec, Oxford, UK) for 90 min at room temperature, followed by secondary detection for 40 min in the dark with a 1:200 dilution of FITC-conjugated, goat antimouse IgG1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Stainings were observed with a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and recorded by a digital camera (Carl Zeiss).

Flow cytometry
After treatment with NaHS, U937 cells were blocked with 2% FBS for 10 min. Fluorescent labeling was performed by incubating the cells with mouse antihuman CD11b mAb (Serotec; 1:25) for 30 min, followed by secondary detection for 30 min in the dark with FITC-tagged, goat antimouse IgG1 (Santa Cruz Biotechnology; 1:200). After staining, cells were analyzed by the CyAnTM ADP flow cytometer (Dako, Glostrup, Denmark).

NF-{kappa}B activity assay
Nuclear fractions from cells were extracted with a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s protocol. Protein concentrations of the nuclear extracts were determined by the Bradford assay using a commercial kit (Quick-StartTM, Bio-Rad). NF-{kappa}B DNA-binding activity was analyzed using the TransAMTM NF-{kappa}B p65 transcription factor assay kit (Active Motif) following the manufacturer’s instructions. Briefly, this kit is based on an ELISA format and is carried out in a 96-well plate with oligonucleotide containing the NF-{kappa}B consensus-binding sequence (5'-GGGACTTTCC-3') immobilized on the wells. Nuclear extracts (5 µg) were incubated in the wells, and bound NF-{kappa}B p65 were then detected by a specific primary antibody. A HRP-conjugated secondary antibody was then used to detect the bound primary antibody and provided the basis for colorimetric quantification. The enzymatic product was measured at 450 nm by a microplate reader (Tecan Systems). To monitor the specificity of this assay, the wild-type or mutated NF-{kappa}B consensus oligonucleotide was added into the competitive or mutated competitive control wells prior to the addition of nuclear extracts.

Western immunoblot
Western blotting was performed with whole cell extracts obtained using radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (Sigma Chemical Co.). Protein concentration was determined by the method of Bradford. Protein samples (50 µg) were separated by SDS-PAGE on Novex® 10% Tris-glycine polyacrylamide gels (Invitrogen) and transferred onto polyvinylidene difluoride membranes (Invitrogen) by electroblotting in Novex® transfer buffer (Invitrogen) containing 20% (v/v) methanol. Membranes were then washed, blocked, and probed overnight at 4°C with rabbit anti-I{kappa}B{alpha}, ERK1/2, phospho-ERK1/2, p38 MAPK, phospho-p38 MAPK, JNK, or phospho-JNK antibodies (Cell Signaling Technology, Danvers, MA, USA; 1:1000 dilution), respectively, followed by secondary detection for 2 h with a 1:2000 dilution of HRP-conjugated, goat antirabbit IgG (Santa Cruz Biotechnology). Membranes were washed and then incubated in SuperSignalTM West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA) before exposure to X-ray films (CL-XPosureTM, Pierce). Gels were calibrated by protein kaleidoscope standards (Bio-Rad). ß-Tubulin (rabbit antibody was purchased from Santa Cruz Biotechnology; 1:3000 dilution) was applied as an internal control to normalize protein loading. The intensity of bands was quantified using LabWorksTM Image Analysis software (UVP).

ERK activity assay
ERK1/2 activity was analyzed using the p44/42 MAPK assay kit (Cell Signaling Technology). Cell lysates were incubated with immobilized phospho-p44/42 antibody bead slurry overnight at 4°C. Then, the immunoprecipitation pellets were incubated in kinase buffer containing Ets-like protein 1 (Elk-1) fusion protein (ERK substrate) and ATP. Finally, the phosphorylated Elk-1 fusion protein was detected by Western blotting using phospho-Elk-1 antibody. The intensity of bands was quantified using LabWorksTM Image Analysis software (UVP).

Statistical analysis
Data are expressed as the mean ± SD. Statistical evaluation was performed by unpaired Student’s t test or when multiple comparisons were made by ANOVA. A P value of <0.05 was considered to indicate a significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of NaHS treatment on the gene expression of several proinflammatory cytokines in U937 cells
We first investigated whether H2S would stimulate the activation of U937 cells, which results in enhancing the expression of some proinflammatory cytokines. Differentiated U937 cells were treated with 0.1 mM or 1 mM of the H2S donor, NaHS, for 0, 2, or 6 h. Then, total RNA from cells was extracted, and RT-PCR for TNF-{alpha}, IL-1ß, and IL-6 was performed. Our results showed that after 2 or 6 h of incubation with 0.1 mM or 1 mM NaHS added to the culture medium, U937 cells displayed significant up-regulation of the mRNA expression of TNF-{alpha}, IL-1ß, and IL-6 (Fig. 1 ).


Figure 1
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  		Figure 1. Induction of TNF-{alpha}, IL-1ß, and IL-6 mRNA expression by H2S in U937 cells. Differentiated U937 cells were treated with 0.1 mM or 1 mM of the H2S donor, NaHS, for 2 or 6 h. The mRNA levels of TNF-{alpha}, IL-1ß, and IL-6 were determined by RT-PCR. Sample loading was normalized with the housekeeping gene ß-actin. The histograms represent the ratios of TNF-{alpha}, IL-1ß, and IL-6 optical density to ß-actin, expressed as fold increases over basal levels (0 h). Data shown are the mean ± SD of the three experiments. *, P < 0.05, compared with basal level (0 h).

 
Effect of NaHS treatment on the secretion of cytokines in U937 cells
Based on the above finding that H2S induced elevation of TNF-{alpha}, IL-1ß, and IL-6 mRNA expression, we checked whether H2S could influence the protein levels of these proinflammatory cytokines. Cells were treated with 0.1 mM or 1 mM NaHS for 0, 2, 4, 6, and 8 h, respectively. The supernatants were harvested at the indicated time-points and were used to measure TNF-{alpha}, IL-1ß, and IL-6 levels by ELISA. Our results showed that NaHS treatment markedly promoted TNF-{alpha}, IL-1ß, and IL-6 production and release from U937 monocytes (Fig. 2 ).


Figure 2
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  		Figure 2. Up-regulation of TNF-{alpha}, IL-1ß, and IL-6 protein production by H2S. Cells were treated with 0.1 mM or 1 mM NaHS for 2, 4, 6, and 8 h, respectively. The concentrations of TNF-{alpha}, IL-1ß, and IL-6 were measured by ELISA with the supernatants harvested at the indicated time-points. Results are expressed as the mean ± SD for triplicate measurements and from three separate experiments. *, P < 0.05, compared with basal level (0 h).

 
Effect of NaHS treatment on the surface expression of CD11b on U937 cells
Subsequently, we used CD11b, a monocyte activation marker, as an indicator to further evaluate the activation of U937 monocytes induced by H2S. Cells were treated with 0.1 mM NaHS for 6 h. Immunofluorescent and flow cytometric analysis revealed that NaHS treatment resulted in a significant up-regulation of the expression of CD11b on the cell surface (Fig. 3 ).


Figure 3
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  		Figure 3. Up-regulation of CD11b expression by H2S. Cells were treated with 0.1 mM NaHS for 6 h. The expression of CD11b on the cell surface was measured by fluorescence microscopy (A and B) and flow cytometry (C). Typical results from three separate experiments are shown. Original magnification, x400 (A); x1000 (B).

 
Effect of NaHS treatment on the activity of NF-{kappa}B p65 in U937 cells
The transcription factor NF-{kappa}B has been believed to play a key role in the signal transduction during human monocyte activation, and all the TNF-{alpha}, IL-1ß, and IL-6 genes are NF-{kappa}B-responsive genes. Therefore, we studied the effect of H2S on the transactivation of NF-{kappa}B in U937 cells, which were treated with NaHS (0.01–1 mM) for 0–120 min. Nuclear fractions from cells were extracted. NF-{kappa}B DNA-binding assay revealed that treatment of NaHS led to a notable increase in the activity of NF-{kappa}B p65, indicating that NF-{kappa}B p65 plays an important role in the signaling pathway of H2S-induced activation of U937 monocytes. Analysis of the kinetics of this induction showed that the DNA-binding activity of NF-{kappa}B p65 was increased after 10 min of incubation with NaHS and reached a peak at 30 min followed by a decrease after 60 min (Fig. 4A ). A dose-dependent experiment showed that the maximum effect was observed at a concentration of 0.1 mM NaHS (Fig. 4B) . Consequently, 0.1 mM NaHS was used for all the subsequent experiments.


Figure 4
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  		Figure 4. H2S stimulates NF-{kappa}B p65 activation. (A) The time course of NF-{kappa}B p65 activation induced by H2S. Cells were treated with NaHS (0.1 or 1 mM) for 0–120 min. Nuclear fractions were extracted and used for NF-{kappa}B DNA-binding assay. Results are the mean ± SD for triplicate measurements and from three separate experiments. *, P < 0.05, compared with basal level (0 min). (B) The dose dependence of H2S-induced NF-{kappa}B p65 activation. Cells were stimulated with 0.01–1 mM NaHS for 30 min. Results are the mean ± SD for triplicate measurements and from three separate experiments. *, P < 0.05, compared with the value in unstimulated cells (Control).

 
In a separate experiment, we cultured the cells overnight with 30 µM NaHS and then incubated them with another 20 µM NaHS for 10, 30, or 60 min. We observed increases of NF-{kappa}B translocation by 18%, 48%, or 29%, respectively, when compared with the control cells (only incubated with 30 µM NaHS overnight).

Effect of NaHS treatment on the level of I{kappa}B{alpha} in U937 cells
I{kappa}B{alpha} is a key intracellular regulator of the activity of NF-{kappa}B. Its degradation can be regarded as a convincing marker of NF-{kappa}B activation. Therefore, the degradation profile of I{kappa}B{alpha} induced by H2S was investigated. Cells were treated with NaHS at a concentration of 0.1 mM for 0–120 min. Western blot analysis performed with the whole cell lysates extracted from these cells revealed that NaHS treatment caused a transient degradation of I{kappa}B{alpha}, with a marginal decrease at 5 min and evident at 10 and 30 min of incubation with NaHS but returning to control level by 60 min (Fig. 5 ). These results corroborated our findings about the kinetics of NF-{kappa}B activation.


Figure 5
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  		Figure 5. H2S induces I{kappa}B{alpha} degradation. Cells were incubated with 0.1 mM NaHS for 0–120 min, and whole cell lysates were examined by Western blot with the anti-I{kappa}B{alpha} antibody. Equal sample loading was determined by internal control ß-tubulin. (A) Results shown are representative blots from four independent experiments. (B) The histogram represents the ratio of I{kappa}B{alpha} OD:ß-tubulin, expressed as percentages of basal level (0 min). Data shown are the mean ± SD of the four experiments. *, P < 0.05, compared with basal level (0 min).

 
Effect of pretreatment of Bay 11-7082, a specific NF-{kappa}B blocker, on the activation of NF-{kappa}B p65 induced by H2S in U937 cells
To study if H2S stimulates U937 cell activation through the NF-{kappa}B signal transduction pathway, we used Bay 11-7082, a selective and irreversible inhibitor of I{kappa}B{alpha} phosphorylation, which disrupts NF-{kappa}B function by sparing I{kappa}B{alpha} from proteasomal degradation, thereby permitting it to bind to and inactivate NF-{kappa}B [34 ]. Cells were preincubated with Bay 11-7082 (5, 10, or 30 µM) for 1 h before treated with NaHS (0.1 mM) for 30 min. Then, nuclear extracts were collected, and NF-{kappa}B assay was performed. As shown in Figure 6A , Bay 11-7082 strongly prevented the transactivation of NF-{kappa}B p65 induced by H2S. This effect was dose-dependent, and 30 µM Bay 11-7082 almost completely abolished NF-{kappa}B p65 activation.


Figure 6
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  		Figure 6. The H2S-induced NF-{kappa}B activation and cytokine production are attenuated by NF-{kappa}B inhibitor Bay 11-7082. (A) Cells were preincubated with Bay 11-7082 (5, 10, or 30 µM) for 1 h before treated with NaHS (0.1 mM) for 30 min. Nuclear extracts were then collected, and NF-{kappa}B assay was performed. (B) Cells were preincubated with 30 µM Bay 11-7082 for 1 h before treated with 0.1 mM NaHS for 8 h. The supernatants were collected and assayed for TNF-{alpha}, IL-1ß, and IL-6 by ELISA. Results are the mean ± SD for triplicate measurements and from three separate experiments. *, P < 0.05, compared with the value in unstimulated cells (Control). #, P < 0.05, compared with the value in cells treated with NaHS alone.

 
Effect of Bay 11-7082 on the production of cytokines induced by H2S in U937 cells
Cells were preincubated with 30 µM Bay 11-7082 for 1 h before treated with 0.1 mM NaHS for 8 h. The supernatants were collected and assayed for TNF-{alpha}, IL-1ß, and IL-6 by ELISA. The results showed that pretreatment with Bay 11-7082 obviously inhibited the NaHS-stimulated secretion of these proinflammatory cytokines from U937 cells (Fig. 6B) , substantiating the important role of NF-{kappa}B in the activation of U937 monocytes caused by H2S.

Effects of NaHS treatment on the phosphorylation of MAPKs and the activity of ERK in U937 cells
To understand further the molecular mechanism of H2S-induced U937 cell activation, lysates obtained at various times from NaHS-treated cells were subjected to Western blot analysis for phosphorylated or total MAPKs. Treatment with NaHS led to a robust up-regulation of the phosphorylation of ERK but not of p38 MAPK and JNK. ERK1/2 phosphorylation appeared and peaked at 3 min incubation with NaHS followed by a rapid decline (Fig. 7 ).


Figure 7
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  		Figure 7. H2S induces phosphorylation of ERK1/2 but not of p38 MAPK and JNK. Cells were incubated with 0.1 mM NaHS for 0–120 min, and whole cell lysates were examined by Western blot with anti-ERK1/2, phospho-ERK1/2 (P-ERK), p38 MAPK, phospho-p38 MAPK, JNK, or phospho-JNK antibodies, respectively. (A) Results shown are representative blots from three independent experiments. (B) The histograms represent the ratios of the optical density of phospho-ERK1/2:total ERK1/2 expressed as percentages of the highest levels (3 min). Data shown are the mean ± SD of the three experiments. *, P < 0.05, compared with basal level (0 min).

 
According to the fact that phosphorylation of MAPKs results in protein conformational change and activation of them [33 ], we next detected the activity of ERK after NaHS treatment. We found that H2S induced a strong but transient activation of ERK1/2 (Fig. 8 ). This result was consistent with our above finding in the phosphorylation of ERK1/2.


Figure 8
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  		Figure 8. H2S stimulates ERK1/2 activation. Cells were incubated with 0.1 mM NaHS for 0–30 min. ERK1/2 activity was determined by immunoprecipitation studies using an Elk-1 fusion protein as a substrate. (A) Result shown is a representative blot from three independent experiments. (B) The histogram represents the optical density of phospho-Elk-1 expressed as percentages of the highest level (5 min). Data shown are the mean ± SD of the three experiments. *, P < 0.05, compared with basal level (0 min).

 
Effect of PD98059, a specific ERK pathway inhibitor, on the H2S-induced NF-{kappa}B p65 activation in U937 cells
To investigate whether ERK is involved in H2S-induced NF-{kappa}B activation, we used PD98059, a potent and highly selective inhibitor of the ERK kinase MEK1. Cells were preincubated with PD98059 (10, 30, 50, or 100 µM) for 1 h before treated with NaHS (0.1 mM) for 30 min. Then, nuclear extracts were collected, and NF-{kappa}B activity assay was performed. As shown in Figure 9 , PD98059 obviously suppressed the H2S exposure-associated increase in NF-{kappa}B p65 activity. This effect was dose-dependent within the range of 10–50 µM, whereas 100 µM PD98059 did not cause further inhibition.


Figure 9
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  		Figure 9. Inhibitory effect of MEK1 antagonist PD98059 on H2S-induced NF-{kappa}B p65 activation. Cells were preincubated with PD98059 (10, 30, 50, or 100 µM) for 1 h before treated with NaHS (0.1 mM) for 30 min. Nuclear fractions were then extracted, and NF-{kappa}B activity assay was performed. Results are the mean ± SD for triplicate measurements and from three separate experiments. *, P < 0.05, compared with the value in unstimulated cells (Control). #, P < 0.05, compared with the value in cells treated with NaHS alone.

 
Effect of glibenclamide, an ATP-sensitive K+ channel (KATP) blocker, on the NF-{kappa}B p65 activation and cytokine production induced by H2S in U937 cells
To elucidate whether KATP channels were involved in H2S-induced monocyte activation, U937 cells were incubated with glibenclamide (10 µM) for 20 min prior to the application of NaHS (0.1 mM). Unfortunately, we did not find any significant reduction in H2S-induced NF-{kappa}B activation or cytokine secretion in the presence of this KATP blocker (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To date, abundant evidence supporting a proinflammatory effect of H2S in vivo has been obtained [11 12 13 , 28 ]. However, to the best of our knowledge, there is little biological information about the mechanism by which endogenous H2S acts as an inflammatory mediator. In the current study, we performed a series of in vitro experiments to investigate the potential effect of H2S on the activation of monocytes using a human monocyte model.

We first investigated the effects of H2S on gene expression and protein production of some cytokines in differentiated U937 cells. We found that treatment with the H2S donor, NaHS, induced significant increases in the mRNA expression and subsequent protein secretion of TNF-{alpha}, IL-1ß, and IL-6 compared with basal levels. TNF-{alpha}, IL-1ß, and IL-6 have all been well known to be multifunctional, proinflammatory cytokines with pivotal roles in the pathogenesis and development of inflammation. Therefore, our findings suggest that H2S takes part in inflammation through, at least partially, the activation of monocytes and production of proinflammatory cytokines by these cells. In addition, as we know, the cell surface expression of CD11b (also designated as integrin {alpha}M or macrophage antigen complex-1{alpha}) is a key parameter of monocyte activation [35 ]. Accordingly, our present result that NaHS treatment led to a significant up-regulation of CD11b expression on U937 cells validates the activation of human monocytes stimulated by H2S.

Although the fact that H2S stimulates human monocyte activation has emerged, the signaling pathway in this process still needs to be determined. In recent years, numerous reports have demonstrated some molecular mechanisms by which H2S plays a part in other biological processes. For example, H2S induces vasorelaxation by a KATP-dependent or an extracellular Ca2+-dependent mechanism [9 , 17 , 18 ] and plays a cardioprotection role by activating protein kinase C (PKC) and KATP channels [22 , 23 ]. H2S induces apoptosis of smooth muscle cells via the activation of MAPKs and caspase-3 [36 , 37 ] and induces apoptosis in pancreatic acinar cells via the mitochondrial pathway as well as the death receptor pathway [38 ]. In astrocytes, H2S increases intracellular Ca2+ and induces Ca2+ waves [39 ]. In addition, H2S triggers intestinal cells to proliferate via a MAPK-dependent mechanism, which results in activation of c-Jun and up-regulation of genes involved in proliferation [40 ]. However, the molecular mechanisms underlying the effects of H2S on immune cells were seldom studied. One recent report mentioned that H2S prevented apoptosis of human primary neutrophils via inhibition of p38 MAPK and caspase-3 [41 ].

In the present study, we have observed that in human U937 monocytes, H2S is able to promote the expression of several cytokines, a process that has been known to be controlled largely by NF-{kappa}B activation, a transcription factor that has crucial modulatory roles in inflammation, immunity, cell proliferation, and apoptosis [32 , 42 ]. It describes various dimeric complexes of members of the Rel protein family, which comprises Rel (c-Rel), RelA (p65), RelB, NF-{kappa}B1 (p50 and its precursor p105), and NF-{kappa}B2 (p52 and its precursor p100) [43 , 44 ]. The members of the family share a conserved region, the Rel homology domain, containing the site-specific, DNA-binding domains as well as the nuclear localization signal (NLS) [45 ]. In resting cells, NF-{kappa}B is retained in the cytoplasm, associating with members of the I{kappa}B family such as I{kappa}B{alpha}, I{kappa}Bß, and I{kappa}B{epsilon}. By interacting with the Rel homology domain, I{kappa}B serves to occlude the DNA-binding domain and NLSs of NF-{kappa}B [46 ]. Extracellular stimuli initiate a signaling cascade, which leads to the phosphorylation of I{kappa}B by I{kappa}B kinases (IKKs), IKK{alpha} and IKKß. The phosphorylated I{kappa}B is then ubiquitinated and degraded rapidly. With the NLSs and DNA-binding domains unmasked, free NF-{kappa}B translocates into the nucleus, where it binds to the {kappa}B sites in the promoters of its target genes and activates the transcription of these genes [47 , 48 ]. Besides this classical activation pathway, NF-{kappa}B can also be activated via two other atypical pathways. One is through the processing of p100 and release of RelB-p52 into the nucleus; another is through the phosphorylation of p65 at multiple serine sites by some protein kinases [42 , 44 ].

In this report, we performed a NF-{kappa}B DNA-binding activity assay as well as Western immunoblotting for I{kappa}B{alpha} to investigate the potential role of NF-{kappa}B in the activation of human U937 monocytes stimulated by H2S. Our results revealed that H2S could induce a rapid degradation of I{kappa}B{alpha}, accompanied with an activation of NF-{kappa}B p65 in U937 cells, and this effect was attenuated by Bay 11-7082, a specific pharmacological inhibitor of NF-{kappa}B. Furthermore, pretreatment of cells with Bay 11-7082 substantially antagonized H2S-triggered cytokine secretion, substantiating that the proinflammatory effects of H2S on human monocytes are mediated by the classical NF-{kappa}B-transduction pathway. In addition, NF-{kappa}B transactivation was not only observed in fresh cells incubated with NaHS but also in those cells cultured with 30 µM NaHS overnight and then incubated with another 20 µM NaHS. This process was more similar to the in vivo state, as in the human body, cells are always in a H2S environment, and the level of H2S increases during inflammation.

Subsequently, we focused our minds on the signaling cascade between H2S stimulation and NF-{kappa}B transactivation in human monocytes. Given the fact that MAPKs mediate some other H2S-initiated physiological or pathological processes, as reported by previous studies [36 , 37 ], and these kinases have been known to be upstream activators of NF-{kappa}B [33 , 44 ], we investigated their functions in H2S-induced NF-{kappa}B activation in U937 cells. The MAPK family represents important signal transduction machinery and occupies a central position in a wide range of cellular responses, including growth, differentiation, apoptosis, and inflammation. The mammalian MAPK family consists of three major members, including ERK, p38, and JNK. Different MAPKs are activated by different stimuli and target at different downstream molecules and therefore, perform different functions [33 , 49 ]. In the present study, we found that NaHS treatment resulted in a rapid and strong activation of ERK1/2 but not of p38 MAPK and JNK, and pretreatment with MEK1 (a kinase of ERK) antagonist PD98059 notably suppressed the H2S-dependent increase in NF-{kappa}B p65 activity. This discovery indicates that ERK is involved in the signal transduction of H2S-stimulated human monocyte activation. The possible pathway is shown in Figure 10 . A more precise cascade remains to be elucidated. The roles of Raf (ERK kinase kinase), PI-3K, and PKC should be worth exploring.


Figure 10
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  		Figure 10. A model for the signaling cascade that mediates H2S-induced U937 cell activation. Treatment of U937 cells by H2S activates ERK activity, which results in I{kappa}B{alpha} degradation with a subsequent increase in NF-{kappa}B-binding activity, leading to increased production of TNF-{alpha}, IL-1ß, and IL-6 and a subsequent proinflammatory response.

 
In addition, as in our results, 30 µM Bay 11-7082 could not completely abolish the up-regulation of cytokine production in U937 cells caused by H2S, whereas it could absolutely abrogate the H2S-stimulated NF-{kappa}B p65 activation, it may be worthwhile to seriously consider that other NF-{kappa}B subunits (e.g., p52, RelB, and others) or transcription factors (e.g., AP-1, STAT, and others) are also involved in the signal transduction of H2S-induced human monocyte activation. In other words, it seems reasonable to extrapolate that besides the NF-{kappa}B-dependent pathway, there are NF-{kappa}B-independent pathways in this process. Moreover, knowing that inducible NO synthase and neurokinin-1 receptor (a primary receptor for substance P) genes are also NF-{kappa}B-responsive genes [50 51 52 ] and that our recent reports have suggested that H2S may work in concert with NO or substance P (SP) to promote inflammatory changes in endotoxic shock or H2S-induced lung injury [53 , 54 ], we postulate that the synergism of H2S and NO or SP is one of the mechanisms by which H2S plays a part in inflammatory responses.

Finally, as the KATP channel had been demonstrated to be involved in H2S-induced vasorelaxation and cardioprotection, a preliminary study was performed to determine whether this K+ channel was also the target of H2S on human monocyte. The H2S-provoked NF-{kappa}B activation and cytokine production in U937 cells was not affected by the KATP blocker glibenclamide, suggesting that KATP channels might not be responsible for the H2S-induced monocyte activation. Further studies can be performed in the future to identify the roles of other types of K+ channels or other ion channels in this process.

In summary, our present findings provide the first evidence that H2S stimulates the activation of human monocytes with the expression and production of proinflammatory cytokines in vitro. Transcription factor NF-{kappa}B and its upstream activator ERK play key roles in this response. The precise signaling pathway involved in this process will be the subject of future studies.


   ACKNOWLEDGEMENTS
 
This work was supported by the Biomedical Research Council (grant no. R-184-000-094-305) and Office of Life Sciences Cardiovascular Biology Program (grant no. R-184-000-074-712), National University of Singapore. We thank Dr. Alirio J. Melendez for providing us U937 cells.

Received October 2, 2006; revised December 29, 2006; accepted January 8, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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