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Originally published online as doi:10.1189/jlb.0407237 on June 28, 2007

Published online before print June 28, 2007
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(Journal of Leukocyte Biology. 2007;82:894-905.)
© 2007 by Society for Leukocyte Biology

Endogenous hydrogen sulfide regulates leukocyte trafficking in cecal ligation and puncture-induced sepsis

Huili Zhang*, Liang Zhi*, Shabbir M. Moochhala{dagger}, Philip Keith Moore* and Madhav Bhatia*,1

* Department of Pharmacology, National University of Singapore, Singapore; and
{dagger} DSO National Laboratories, Singapore

1 Correspondence: Cardiovascular Biology Research Programme, Department of Pharmacology, Centre for Life Sciences, National University of Singapore, 28 Medical Drive, #03-02, Singapore 117456. E-mail: mbhatia{at}nus.edu.sg


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hydrogen sulfide (H2S) is recognized increasingly as a proinflammatory mediator in various inflammatory conditions. Here, we have investigated the role of H2S in regulating expression of some endothelial adhesion molecules and recruitment of leukocytes to inflamed sites in sepsis. Male Swiss mice were subjected to cecal ligation and puncture (CLP)-induced sepsis and treated with saline (i.p.), DL-propargylglycine (PAG; 50 mg/kg, i.p.), an inhibitor of H2S formation or NaHS (10 mg/kg, i.p.), an H2S donor. PAG was administered 1 h before or after the induction of sepsis, and NaHS was given at the same time of CLP. Using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of PAG reduced leukocyte rolling and adherence significantly in mesenteric venules coupled with decreased mRNA and protein levels of adhesion molecules (ICAM-1, P-selectin, and E-selectin) in lung and liver. In contrast, injection of NaHS up-regulated leukocyte rolling and attachment significantly, as well as tissue levels of adhesion molecules in sepsis. Conversely, normal mice were given NaHS (10 mg/kg, i.p.) to induce lung inflammation, with or without NF-{kappa}B inhibitor BAY 11-7082 pretreatment. NaHS treatment enhanced the level of adhesion molecules and neutrophil infiltration in lung. These alterations were reversed by pretreatment with BAY 11-7082. Moreover, expression of CXCR2 in neutrophils obtained from H2S-treated mice was up-regulated significantly, leading to an obvious elevation in MIP-2-directed migration of neutrophils. Therefore, H2S acts as an important endogenous regulator of leukocyte activation and trafficking during an inflammatory response.

Key Words: inflammation • adhesion molecules • leukocyte–endothelial interaction • DL-propargylglycine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial cell-directed recruitment and activation of neutrophils at the site of infection are essential features of the innate immune response to infection [1 ]. Leukocyte infiltration from blood vessel to tissue is a multistep process, which includes initial interaction between leukocytes and endothelium, followed by transient weak adhesion (characterized by leukocyte rolling) and firm leukocyte attachment to blood vessel walls [2 ]. Firm attachment then allows leukocytes to migrate across the surface of endothelial cells into the target sites. Central to this process is the up-regulation of complementary adhesion molecules and ligands on neutrophils and endothelium induced by bacterial or proinflammatory mediators [2 ]. Chemotactic factors and their receptors also play a pivotal role in regulating the activation and movements of leukocytes through the extracellular matrix [3 ].

Sepsis remains a common and serious medical condition, frequently occurring after hemorrhage, trauma, burn, or abdominal surgery. It is caused by a severe systemic infection resulting in a systemic inflammatory response. Many of the adverse outcomes of severe sepsis can be traced to an abnormally enhanced inflammatory response. For example, widespread up-regulation of adhesion molecules by excessive proinflammatory mediators results in rolling and adherence of leukocytes to endothelial cells in infected and noninfected tissues [3 , 4 ]. Although leukocyte activation and adhesion to endothelium play an important role in host defense and repair of tissue damage, it may contribute to tissue damage and multiple organ dysfunction under some conditions [4 ]. Therefore, it is important to explore the regulation of leukocyte trafficking in sepsis.

Hydrogen sulfide (H2S) has been well known for several decades as a toxic gas with the smell of rotten eggs. However, it is also generated endogenously during cysteine metabolism in many types of mammalian cells in the reaction catalyzed by cystathionine ß-synthase (EC4.2.1.22) and cystathionine {gamma}-lyase (CSE; EC4.4.1.1) [5 , 6 ]. Recent findings have suggested that endogenous H2S is associated with inflammation. For example, an obvious increase in H2S generation and up-regulation of CSE activity were seen in animal models of hindpaw edema, acute pancreatitis, endotoxemia, and sepsis, whereas inhibition of H2S formation alleviated the severity of pancreatitis and sepsis [7 8 9 10 ]. However, the role of H2S in regulating leukocyte recruitment in inflammation remains conflicting. Early data showed that the chemotaxis and recruitment of polymorphonuclear cells (PMN) were inhibited to some extent by in vitro exposure to sodium sulfide, an H2S donor [11 , 12 ]. Another in vitro study indicated that PMN were able to function in infected sites with high sulfide levels [13 ]. Recently, it has been shown that H2S donors (NaHS and Na2S) inhibited aspirin-induced leukocyte rolling and adherence in mesenteric venules [14 , 15 ].

In light of these findings, the aim of this study was to investigate the role of endogenous H2S in regulating leukocyte–endothelium interaction and its relevance to tissue levels of adhesion molecules in cecal ligation and puncture (CLP)-induced sepsis. We also examined the effect of H2S on infiltration and chemotactic activity of leukocyte in H2S-induced lung injury. Our data suggest that H2S functions as an endogenous regulator of the leukocyte–endothelium system and promotes the recruitment of PMN to the target sites in inflamed tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Induction of sepsis
All experiments were approved by the animal ethics committee of National University of Singapore and carried out in accordance with the established Guiding Principles for Animal Research. The model of CLP-induced sepsis described previously was used with minor modifications [16 ]. Male Swiss albino mice (25–30 g) were anesthetized lightly with the mixture of ketamine and medetomindine [0.75 ml ketamine (100 mg/ml) and 1 ml medetomindine (1 mg/ml), dissolved in 8.25 ml distilled water; 7.5 ml/kg] under aseptic conditions. After shaving the abdominal fur and applying a topical disinfectant, a small, midline incision was made through the skin and peritoneum of the abdomen to expose the cecum. The cecal appendage was ligated 3–5 mm below the ileocecal valve with Silkam 4/0 thread without occluding the bowel passage and then perforated in two locations with a 22-gauge needle distal to the point of ligation. After this, a small amount of stool was squeezed out through both the holes. Finally, the bowel was repositioned, and the abdomen was stitched up with sterile Permilene 5/0 thread. Animals with sham operation underwent the same procedure without CLP.

DL-Propargylglycine (PAG; Sigma Chemical Co., St. Louis, MO, USA, 50 mg/kg, i.p.), an irreversible inhibitor of CSE or saline, was administered 1 h before ("prophylactic") or 1 h after ("therapeutic") the CLP or sham operation [7 , 17 ]. In the NaHS intervention experiment, mice underwent CLP operation and were simultaneously given NaHS (Sigma Chemical Co.; 10 mg/kg, i.p.) or saline. Eight hours after the operation, animals were killed by an i.p. injection of a lethal dose of pentobarbitone (90 mg/kg). Samples of lung and liver were collected and stored at –80°C for subsequent measurement by RT-PCR and ELISA.

Time course study after CLP challenge
Mice were divided into two groups and treated with CLP or sham operation (n=10–12 in each group). Mice were killed 4, 8, 16, and 24 h after CLP or sham operation, and the samples of lung and liver were collected and stored at –80°C for measurement of tissue myeloperoxidase (MPO) activity.

Induction of lung injury by H2S
Male Swiss albino mice (25–30 g) were i.p.-injected with NaHS (10 mg/kg) or saline. BAY 11-7082 (Calbiochem, San Diego, CA, USA), an inhibitor of NF-{kappa}B, was dissolved in 1% DMSO and i.p.-administered to mice at doses of 5, 10, and 20 mg/kg, 30 min before injection of NaHS. Some mice were given antileukinate (52.6 mg/kg, s.c.), a CXCR2 antagonist, 30 min before injection of saline or NaHS [18 ]. Control animals were only injected with vehicle (1% DMSO or saline), 30 min before injection of saline or NaHS. One hour after injection of NaHS, animals were killed by an i.p. injection of a lethal dose of pentobarbitone (90 mg/kg). Samples of lung were harvested and stored at –80°C for subsequent measurement of MPO activity and adhesion molecules.

Intravital microscopy
The microscopic setup used has been described in detail previously [19 , 20 ]. Male Swiss mice (25–30 g) were anesthetized initially with the mixture of ketamine and medetomindine [0.75 ml ketamine (100 mg/ml) and 1 ml medetomindine (1 mg/ml), dissolved in 8.25 ml distilled water; 7.5 ml/kg]. A midline, abdominal incision was made, and the animals were placed in a supine position. A segment of the mid-jejunum was exteriorized through the abdominal incision, and all exposed tissue was covered with saline-soaked gauze to minimize tissue dehydration. The mesentery was placed carefully over a water chamber. The temperature of the chamber was maintained at 37°C with a constant temperature circulator. The mesentery was suffused with warm, bicarbonate-buffered saline (pH 7.4). An intravital microscope (Nikon, Japan) with a x20 objective lens and a x10 eyepiece was used to observe the mesenteric microcirculation. For visualization of leukocytes, the fluorescent marker rhodamine 6G (Sigma Chemical Co.) was injected i.v. as a single bolus of 0.15 mg/kg body weight, immediately prior to each measurement [19 ]. Epi-illumination was achieved with a variable HBO mercury lamp. A video camera mounted on the microscope projected the image onto a color monitor, and the images were recorded for playback analysis. Three to five venules (40–50 mm in diameter) were selected in each experiment, and to minimize variability, the same section of each venule was observed throughout the experiment. Data from individual vessels were averaged for each mouse, and the group mean was generated from these averages. n was used to denote the number of mice examined. Venular diameter was measured on-line using a video caliper. The number of adherent and rolling leukocytes was determined off-line during playback of video images, using five different fields for each animal to avoid variability as a result of sampling.

The interaction of leukocytes with the luminal surface of the venular endothelium was investigated 8 h after CLP surgery. Leukocytes were considered adherent to venular endothelium if they remained stationary for 30 s or more within a given 100-µm vessel segment. Rolling leukocytes were defined as those leukocytes, which moved at a velocity less than that of erythrocytes in the same vessel. The number of rolling leukocytes was determined at 1 min intervals. Leukocyte rolling velocity was determined from the time required for a leukocyte to traverse a given distance along the length of a venule.

Semiquantitative RT-PCR analysis of lung and liver adhesion molecule mRNA
Total RNA from lung and liver was extracted with Trizol® reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. Total RNA from PMN was extracted with an RNeasy kit (Qiagen, Germany), according to the manufacturer's instruction. RNA (1 µg) was transcribed reversely using the iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) at 25°C for 5 min, 42°C for 30 min, followed by 85°C for 5 min. The cDNA was used as a template for PCR amplification by iQTM Supermix (Bio-Rad). The primer sequences for detection of ICAM-1, P-selectin, E-selectin, CXCR2, 18S, optimal annealing temperature, optimal cycles, and product sizes were as shown in Table 1 . PCR amplification was carried out in MyCyclerTM (Bio-Rad). The reaction mixture was first subjected to 95°C for 3 min, followed by an optimal cycle of amplifications, consisting of 95°C for 50 s and optimal annealing temperature for 50 s and 72°C for 1 min. PCR products were analyzed on 1.5% w/v agarose gels containing 0.5 µg/ml ethidium bromide.


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  		Table 1. PCR Primer Sequences, Optimal Conditions, and Product Sizes

 
Measurement of adhesion molecules
For the measurement of adhesion molecules (ICAM-1, P-selectin, and E-selectin) in homogenized liver and lung, ELISA kits from R&D Systems (Minneapolis, MN, USA) were used, according to the manufacturer's instructions. The lower limits of detection of ICAM-1, P-selectin, and E-selectin levels were 62.5 pg/ml, 31.25 pg/ml, and 31.25 pg/ml, respectively. The ELISA assay for the three adhesion molecules gave reproducible results with inter-assay variability of less than 9.5% and intra-assay variability of less than 6.5%. Results were then corrected for the DNA content of the tissue samples and were expressed as pg/µg DNA [21 ].

Measurement of MPO
Neutrophil sequestration in lung and liver was quantified by measuring tissue MPO activity. Tissue samples were thawed, homogenized in 20 mM phosphate buffer (pH 7.4), and centrifuged (13,000 g, 10 min, 4°C), and the resulting pellet was resuspended in 50 mM phosphate buffer (pH 6.0) containing 0.5% w/v hexadecyltrimethylammonium bromide (Sigma Chemical Co.). The suspension was subjected to four cycles of freezing and thawing and disrupted further by sonication (40 s). The sample was then centrifuged (13,000 g, 5 min, 4°C), and the supernatant was used for the MPO assay. The reaction mixture consisted of the supernatant (50 µl), 1.6 mM tetramethylbenzidine (Sigma Chemical Co.), 80 mM sodium phosphate buffer (pH 5.4), and 0.3 mM hydrogen peroxide (reagent volume, 50 µl). This mixture was incubated at 37°C for 110 s, the reaction terminated with 50 µl 0.18 M H2SO4, and the absorbance measured at 405 nm. This absorbance was then corrected for the DNA content of the tissue sample, and results are expressed as enzyme activity [21 ].

Blood collection and white blood cell separation
PMN and peripheral blood mononuclear cells (PBMC) were isolated according to the method of Boyum [22 ]. Briefly, heparinized blood was collected by cardiac puncture. Heparinized peripheral blood was diluted 1:2.5 in PBS, layered on top of Ficoll-Paque Plus (Amersham Biosciences, Sweden), and centrifuged at 400 g for 30 min at 18–20°C. PBMC sediment to the plasma-Ficoll Paque interface. Mononuclear cells were collected from a hazy band just above the interface of the Ficoll solution and plasma. The isolated mononuclear cells were washed twice by PBS and resuspended in RPMI-1640 medium. RBC pellet contains PMN. The remaining plasma and Ficoll-Paque were aspirated down to the RBC pellet. A hypotonic lysis of RBC was carried out by adding ice-cold, distilled water to each tube. After gently mixing (45 s), a hypertonic solution (10x PBS) was added to restore isotonicity. After centrifugation (400 g, 6 min at 18–20°C), the supernatant was removed. The lysis was then repeated twice, and the cell pellet (PMN) was resuspended in RPMI-1640 medium. Immediately after isolation, using Turk's staining and trypan blue exclusion, PMN and PBMC were examined routinely for purity and viability, which were >95% and >98%, respectively. The cells were counted using a hemocytometer and light microscope.

Chemotaxis assay
Chemotaxis was evaluated with a QCMTM chemotaxis 96-well cell migration assay (Chemicon, El Segundo, CA, USA). Serum-free medium or chemoattractant solution (150 µL) was added to the lower chamber, and 100 µL cell suspension (2x106/ml) was added to the upper compartment. The cell migration plate assembly was incubated at 37°C with 5% CO2 for 1–2 h. Migratory cells on the bottom of the insert membrane were dissociated from the membrane when incubated with cell detachment buffer. The cells that migrated into the medium in the lower chamber or detached from the membrane by cell detachment buffer were lysed and detected by CyQuant GR dye subsequently. This green fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids. The intensity of fluorescence, which correlated with the number of migrated cells, was measured with a fluorescence plate reader using a 485/520-nm filter set. Results were expressed as fluorescence intensity, relative fluorescence unit (RFU), by subtracting background fluorescence in cells migrating to medium without chemoattractant from specific fluorescence in cells migrating to MIP-2.

Flow cytometric analysis
After fixation by 3.7% formaldehyde at room temperature for 10 min, mouse blood neutrophils (1x106) were washed twice with PBS and then stained with rabbit anti-CXCR2 antibody (dilution 1:100, FabGennix Inc. International, Frisco, TX, USA) for 40 min at room temperature. These cells were washed twice with PBS and then treated with secondary antibody, Alexa Fluor® 488 chicken anti-rabbit IgG (dilution 1:200, Invitrogen), for 30 min at room temperature. The samples were washed extensively twice with PBS and then analyzed with CyanTM ADP flow cytometer (Dako, Carpinteria, CA, USA). Control cases used to determine background fluorescence were samples treated with secondary antibody only. Data acquisition and analysis were carried out using Summit V4.2 software (Dako).

Statistics
The data were expressed as mean ± SEM. The significance of differences among groups was evaluated by ANOVA with post-hoc Tukey's test when comparing three or more groups. The significance of differences between two groups was evaluated by t-test. A P < 0.05 was regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Time course study of MPO activity in CLP-induced sepsis
Changes over time of neutrophil infiltration in lung and liver were studied after onset of sepsis. MPO activity, a marker of PMN infiltration, in lung and liver increased in a time-dependent manner and reached its peak 8 h after CLP (Fig. 1A and 1B ; P<0.05 at each time-point). After 8 h, lung and liver MPO activity in CLP-operated mice started to decline but was still higher than that in sham-operated mice (P<0.05, compared with sham operation). As alteration in tissue MPO activity reached its peak 8 h after CLP challenge, the effect of endogenous H2S on leukocyte trafficking was evaluated at this time-point.


Figure 1
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  		Figure 1. Time course study of lung (A) and liver (B) MPO activity in CLP-induced sepsis. Male Swiss mice were subjected to CLP or sham operation. At indicated time-points (0, 4, 8, 16, 24 h after CLP), mice were killed by an i.p. injection of a lethal dose of pentobarbitone. Results shown are the mean ± SEM (n=10–12 animals in each group). *, P < 0.05, when mice subjected to sham operation were compared with normal mice; **, P < 0.01, when mice subjected to sham operation compared with normal mice; {dagger}, P < 0.05, when mice subjected to CLP were compared with normal mice; {ddagger}, P < 0.01, when mice subjected to CLP were compared with normal mice; #, P < 0.05, when septic mice were compared with sham-operated mice; $, P < 0.01, when septic mice were compared with sham-operated mice.

 
Effect of H2S on leukocyte–endothelium interaction (rolling and adhesion) in CLP-induced sepsis
The microvascular postcapillary venule is the site of leukocyte adhesion and emigration. Thus, we used intravital microcopy to examine the leukocyte–endothelium interaction (rolling and adhesion) in the mesenteric postcapillary venules with resting diameters ranging from 40 to 50 µm. As shown in Figure 2 , CLP-induced sepsis resulted in a significant increase in the number of rolling and adherent cells and a significant decrease in the rolling velocity in comparison with sham operation (P<0.01).


Figure 2
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  		Figure 2. Effect of PAG administration on leukocyte rolling and adhesion to mesenteric venules in septic mice. Bars show the number of rolling leukocytes (A), rolling velocity (B), and adherent leukocytes (C) in postcapillary venules of mesentery, using an in vivo, intravital microscopy assay. Mice with CLP-induced sepsis were given PAG (50 mg/kg, i.p.) or saline randomly, 1 h before (PAG+CLP or saline+CLP) or 1 h after (CLP+PAG or CLP+saline) CLP operation. Sham-operated mice served as controls. The parameters were evaluated 8 h after the surgery. Results shown are the mean ± SEM (n=5 animals in each group). **, Significant difference (P<0.01) between mice subjected to CLP with saline injection and those with sham operation; {ddagger}, significant difference (P<0.01) when PAG-treated animals were compared with saline-treated animals.

 
We used PAG, an irreversible inhibitor of CSE (50 mg/kg, i.p.), to block the formation of endogenous H2S. Treatment of septic mice with PAG, 1 h before or 1 h after CLP, inhibited the leukocyte–endothelium interaction significantly, as evidenced by the reduction in the number of rolling and adherent cells and elevation in the rolling velocity (Fig. 2 ; P<0.01). Conversely, injection of exogenous H2S at the time of CLP operation caused a pronounced and further rise in leukocyte rolling and adhesion compared with saline injection in septic mice (Fig. 3 ; P<0.05).


Figure 3
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  		Figure 3. Effect of NaHS on leukocytes rolling and adherence to mesenteric venules in septic mice. Bars show the number of rolling leukocyte (A), rolling velocity (B), and adherent leukocytes (C) in postcapillary venules of mesentery, using an in vivo, intravital microscopy assay. Mice with CLP-induced sepsis were given NaHS (10 mg/kg, i.p.) or saline randomly at the same time of CLP operation. Normal and sham-operated mice served as controls. The parameters were evaluated 8 h after the surgery. Results shown are the mean ± SEM (n=5 animals in each group). *, Significant difference (P<0.05) when septic mice with saline injection were compared with normal mice or animals with sham operation; **, significant difference (P<0.01) when septic mice with saline injection were compared with normal mice or animals with sham operation; {dagger}, P < 0.05, when NaHS-treated animals were compared with saline-treated animals.

 
Effect of H2S on mRNA and protein expression level of adhesion molecules in CLP-induced sepsis
Leukocyte–endothelium interaction typically involves the action of three different adhesion molecule families: the selectins, integrins, and Igs. Thus, the effect of H2S on mRNA and protein expression levels of adhesion molecules in lung and liver in sepsis were tested. Figures 4 and 5 showed that mRNA and protein levels of adhesion molecules, including ICAM-1, P-selectin, and E-selectin, in lung and liver were elevated significantly in septic mice compared with sham-operated mice (P<0.05). Inhibition of H2S formation by PAG pretreatment or post-treatment reduced the mRNA level and production of ICAM-1, P-selectin, and E-selectin greatly in lung and liver (Figs. 4 and 5 ; P<0.05, compared with saline treatment). In contrast, application of the H2S donor, NaHS, resulted in a significant increase in tissue mRNA and protein levels of ICAM-1, P-selectin, and E-selectin in septic mice (Figs. 6 and 7 ; P<0.05).


Figure 4
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  		Figure 4. Effect of PAG administration on the mRNA expression levels of adhesion molecules (ICAM-1, P-selectin, and E-selectin) in lung (A–C) and liver (D–F) from mice with CLP-induced sepsis. Mice with CLP-induced sepsis were given PAG (50 mg/kg, i.p.) or saline randomly, 1 h before (PAG+CLP or saline+CLP) or 1 h after (CLP+PAG or CLP+saline) CLP operation. Eight hours after CLP or sham operation, the levels of adhesion molecule mRNA (ICAM-1, P-selectin, and E-selectin) in lung and liver were measured by RT-PCR (determined as ratio of band densities of adhesion molecules:18S), as described in Materials and Methods. Mouse 18S served as a control. Results shown are the mean ± SEM (n=12 animals in each group). **, Significant difference (P<0.01) between mice subjected to CLP with saline injection and those with sham operation; {dagger}, P < 0.05, when PAG-treated animals were compared with saline-treated animals; {ddagger}, P < 0.01, when PAG-treated animals were compared with saline-treated animals.

 

Figure 5
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  		Figure 5. Effect of PAG administration on the levels of adhesion molecules (ICAM-1, P-selectin, and E-selectin) in lung (A–C) and liver (D–F) from mice with CLP-induced sepsis. Mice with CLP-induced sepsis were given PAG (50 mg/kg, i.p.) or saline randomly, 1 h before (PAG+CLP or saline+CLP) or 1 h after (CLP+PAG or CLP+saline) CLP operation. Eight hours after CLP or sham operation, the levels of adhesion molecules (ICAM-1, P-selectin, and E-selectin) in lung and liver were measured as described in Materials and Methods. Results shown are the mean ± SEM (n=12 animals in each group). *, Significant difference (P<0.05) between mice subjected to CLP with saline injection and those with sham operation; **, P < 0.01, when mice subjected to CLP with saline injection were compared with animals with sham operation; {dagger}, P < 0.05, when PAG-treated animals were compared with saline-treated animals; {ddagger}, P < 0.01, when PAG-treated animals were compared with saline-treated animals.

 

Figure 6
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  		Figure 6. Effect of NaHS administration on the mRNA expression levels of adhesion molecules (ICAM-1, P-selectin, and E-selectin) in lung (A–C) and liver (D–F) from mice with CLP-induced sepsis. Mice with CLP-induced sepsis were given NaHS (10 mg/kg, i.p.) or saline randomly at the time of onset of CLP. Eight hours after CLP or sham operation, the levels of adhesion molecule mRNA (ICAM-1, P-selectin, E-selectin) in lung and liver were measured by RT-PCR (determined as ratio of band densities of adhesion molecules:18S), as described in Materials and Methods. Mouse 18S served as a control. Results shown are the mean ± SEM (n=12 animals in each group). #, Significant difference (P<0.01) between normal mice and mice with sham operation; *, significant difference when septic mice with saline injection were compared with normal mice (P<0.01) or animals with sham operation (P<0.05); **, significant difference (P<0.01) when septic mice with saline injection were compared with normal mice or animals with sham operation; {dagger}, P < 0.05, when NaHS-treated animals were compared with saline-treated animals; {ddagger}, P < 0.01, when NaHS-treated animals were compared with saline-treated animals.

 

Figure 7
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  		Figure 7. Effect of NaHS administration on the levels of adhesion molecules (ICAM-1, P-selectin, and E-selectin) in lung (A–C) and liver (D–F) from mice with CLP-induced sepsis. Mice with CLP-induced sepsis were given NaHS (10 mg/kg, i.p.) or saline randomly at the time of onset of CLP. Eight hours after CLP or sham operation, the levels of adhesion molecules (ICAM-1, P-selectin, and E-selectin) in lung and liver were measured as described in Materials and Methods. Results shown are the mean ± SEM (n=12 animals in each group). #, Significant difference (P<0.01) between normal mice and mice with sham operation; *, significant difference when septic mice with saline injection were compared with normal mice (P<0.01) or animals with sham operation (P<0.05); **, significant difference (P<0.01) when septic mice with saline injection were compared with normal mice or animals with sham operation; {dagger}, P < 0.05, when NaHS-treated animals were compared with saline-treated animals; {ddagger}, P < 0.01, when NaHS-treated animals were compared with saline-treated animals.

 
Effect of H2S on PMN recruitment in H2S-induced lung inflammation
To investigate the effect of H2S on PMN recruitment and tissue level of adhesion molecules directly, we used an animal model of H2S-induced lung injury. Administration of NaHS (10 mg/kg) in normal mice causes some inflammation in lung, as evidenced by an increase in lung MPO activity, pulmonary level of cytokines, and histological damage [23 , 24 ]. This animal model is also a straightforward way to investigate the function of H2S in inflammation [23 , 24 ]. Neutrophil recruitment was assessed by lung MPO activity and chemotaxis of PMN. Induction of lung inflammation by injection of NaHS (10 mg/kg, i.p.) into normal mice increased neutrophil infiltration (MPO activity) significantly, coupled with elevation in the level of ICAM-1, P-selectin, and E-selectin in lung (Fig. 8A 8B 8C 8D ; P<0.05). This increase was reversed significantly by pretreatment with BAY 11-7082, a selective inhibitor of NF-{kappa}B, in a dose-dependent manner (Fig. 8A 8B 8C 8D ; P<0.05) [25 , 26 ].


Figure 8
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  		Figure 8. Effect of NaHS on the levels of adhesion molecules (A–C) and MPO activity (D) in lung from H2S-treated mice, which were given saline or NaHS (10 mg/kg, i.p.) randomly. NF-{kappa}B inhibitor, BAY 11-7082, or vehicle (1% DMSO, i.p.) were administered to mice at doses of 5, 10, and 20 mg/kg, i.p., 30 min before injection of NaHS. One hour after injection of NaHS, pulmonary levels of adhesion molecules (ICAM-1, P-selectin, and E-selectin) and lung MPO activity were measured as described in Materials and Methods. Results shown are the mean ± SEM (n=10 animals in each group). *, Significant difference (P<0.05) between mice with saline injection and those with NaHS injection; **, significant difference (P<0.01) between mice with saline injection and those with NaHS injection; {dagger}, P < 0.05, when NaHS-treated mice with BAY 11-7082 pretreatment were compared with NaHS-treated mice with vehicle pretreatment; {ddagger}, P < 0.01, when NaHS-treated mice with BAY 11-7082 pretreatment were compared with NaHS-treated mice with vehicle pretreatment.

 
Consistently, chemotactic activity of PMN cells isolated from mice with H2S-induced lung inflammation was enhanced significantly (Fig. 9A ). MIP-2, at various concentrations, attracted more PMN cells obtained from H2S-treated mice than those from normal mice (Fig. 9A , P<0.05). As chemotaxis of PMN requires signaling via CXCR2, next, we investigated the expression of CXCR2 on PMN. A moderate rise in CXCR2 mRNA level was observed in PMN isolated from NaHS-treated mice (Fig. 9B , P<0.05). Flow cytometric analysis also showed that CXCR2 expression on PMN was up-regulated in NaHS-treated mice as compared with normal mice (Fig. 9C and 9D) . In addition, blockage of CXCR2 by pretreatment with antileukinate (52.6 mg/kg, s.c.), a potent inhibitor of binding of CXC chemokines to the receptor (CXCR2), reduced the amplified chemotactic activity of PMN in H2S-induced lung injury (Fig. 9E ; P<0.05) [18 ].


Figure 9
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  		Figure 9. Effect of NaHS on the chemotactic activity of PMN toward MIP-2. (A) Mice were given saline or NaHS (10 mg/kg, i.p.) randomly. One hour after injection of NaHS, PMN were isolated for measurement of chemotactic activity toward various concentrations of MIP-2 (A), the level of CXCR2 mRNA by RT-PCR (B), and the expression of CXCR2 by flow cytometery (C, D). (E) Antileukinate (52.6 mg/kg, i.p.) or vehicle was administered to mice 30 min before injection of NaHS. One hour after injection of NaHS or saline, PMN were isolated for measurement of chemotactic activity toward MIP-2 at a concentration of 25 ng/ml. Chemotactic activity is expressed as fluorescence intensity, in RFU. Results shown are the mean ± SEM (n=6–8 animals in each group). *, Significant difference (P<0.05) between mice with saline injection and those with NaHS injection; **, significant difference (P<0.01) between mice with saline injection and those with NaHS injection; {dagger}, P < 0.05, when NaHS-treated mice with antileukinate pretreatment were compared with NaHS-treated mice with vehicle pretreatment.

 
Finally, to evaluate whether H2S itself could function as a chemoattractant mediator and attract leukocyte directly, we isolated PMN and PBMC from normal mice and observed their migration toward H2S. We added NaHS solution at various concentrations ranging from 10 µM to 500 µM to the lower chamber and incubated the cell migration plate assembly for 1–4 h. NaHS, at lower or higher concentration, could not direct the migration of PMN and PBMC from the upper chamber to the lower chamber (data not shown). This experiment ruled out the possibility that H2S acted as a chemotactic factor in inflammation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Leukocyte migration to infection loci is extremely important for the local control of bacterial growth and consequently, for the prevention of bacterial dissemination. It is mediated by the up-regulation of adhesion molecules and ligands on leukocyte and endothelial cells induced by bacterial products and proinflammatory mediators [4 ]. As a potent vasodilator and atypical neurotransmitter, H2S has been implicated to play a proinflammatory role in systemic inflammation, such as endotoxemia and sepsis [9 , 10 ]. However, there is little information about its potential role in regulating leukocyte recruitment.

Some studies found that the presence of sulfide (1–2 mM), a H2S donor, suppressed the migration of PMN slightly [12 ], whereas Claesson et al. [13 ] reported that H2S had a limited, detrimental effect on the function of PMN. However, these early studies were carried out in vitro, and their results are conflicting. Recently, some in vivo experiments indicated that H2S donors attenuated nonsteroidal anti-inflammatory drug (NSAID)-induced gastric granulocyte infiltration and leukocyte adherence in mesenteric venules [14 , 15 ]. Leukocyte infiltration in an air-pouch model was also suppressed by NaHS [14 ]. These in vivo studies provide evidence suggesting the ability of H2S to regulate inflammatory events occurring at a leukocyte–endothelium interface. Unfortunately, the investigators only examined the involvement of H2S in local inflammation, such as a NSAID-induced gastrointestinal injury and air-punch model. To the best of our knowledge, the present study explored for the first time that in sepsis-associated, systemic inflammation, endogenous H2S may function as a modulator of leukocyte recruitment.

Some studies indicate that leukocyte migration is associated with the severity of CLP-induced sepsis, which correlates with the number of punctures in the cecum [27 , 28 ]. For example, mice with sublethal CLP (two punctures with a 24-G needle) exhibited increased neutrophil migration to the inflammatory sites, whereas mice with lethal CLP (14 punctures with a 21-G needle) presented with impaired neutrophil migration into a peritoneal cavity, suggesting leukopenia in very severe sepsis [28 ]. In the present study, using intravital microscopy, we found that CLP-induced sepsis (two punctures with a 22-G needle) resulted in an obvious increase in leukocyte–endothelial interaction in mesentery venules. Furthermore, an increased neutrophil count in circulation but not leukopenia was reported in our previous study [29 ]. All these findings are consistent with the observations in other studies [28 ]. Conversely, we have shown that inhibition of H2S formation by PAG decreased leukocyte rolling and adhesion in sepsis significantly. Administration of the H2S donor, NaHS, caused an obvious and further rise in leukocyte rolling and adhesion in septic mice. Consistently, our previous data also implicated that H2S contributes to neutrophil infiltration (MPO activity) in inflamed tissues (lung and liver) in sepsis [10 ]. Therefore, H2S, as a proinflammatory mediator, plays an important role in regulating leukocyte–endothelium interaction in systemic inflammation.

Leukocyte trafficking across the endothelium to an extravascular site of infection is typically mediated by three adhesion molecule families: the selectin, intergrins, and Igs. We then examined the correlation between endogenous H2S and adhesion molecules (ICAM-1, P-selectin, and E-selectin). In sepsis, the mRNA and protein expression levels of adhesion molecules in lung and liver were enhanced further by administration of NaHS, whereas their levels were reduced by inhibition of H2S formation. The changes in the tissue levels of adhesion molecules are consistent with those observations in leukocyte rolling and adhesion. Taking these findings into account, it is reasonable to conclude that endogenous H2S may exert its effect at the leukocyte–endothelium interface in sepsis via modulating tissue levels of adhesion molecules. Moreover, our previous experiments showed that H2S regulates the activity of NF-{kappa}B in lung and liver, therefore contributing to the overproduction of proinflammatory mediators in sepsis [24 ]. Therefore, these observations provide direct evidence that H2S regulates leukocyte–endothelium interaction through a mechanism involving NF-{kappa}B activation. The underlying cellular signaling pathway by which H2S modulates the leukocyte trafficking via activation of NF-{kappa}B in sepsis will be the subject of future studies. The latest studies offer an original possibility that H2S may modulate leukocyte rolling and adhesion in NSAID-induced gastric injury via the activation of K+ATP [14 , 15 ].

To ascertain the role of H2S in modulating leukocyte recruitment once again, we performed some experiments in an animal model of H2S-induced lung injury. Application of exogenous H2S in normal mice caused an evident increase in the pulmonary level of adhesion molecules and consequently amplified neutrophil sequestration in the lung. Administration of NaHS in normal mice also induces activation of NF-{kappa}B in the lung [24 ]. As a result, inhibition of NF-{kappa}B activity by BAY 11-7082 greatly reduced up-regulation of adhesion molecules and PMN infiltration induced by exogenous H2S. Taken together, these results reinforce the concept that H2S accounts for the regulation of leukocyte trafficking via activation of NF-{kappa}B and consequent expression of adhesion molecules.

Conversely, our study demonstrated that H2S itself did not possess leukocyte chemoattractant ability. H2S at low and high concentrations cannot direct PMN and PBMC to migrate toward target sites. Although H2S itself cannot attract PMN, it up-regulated the expression of CXCR2 on PMN and thus facilitated MIP-2-directed migration of PMN. Blockage of CXCR2 by pretreatment with antileukinate significantly abolished the augmented chemotactic response of PMN induced by H2S. Unfortunately, the precise mechanism by which H2S stimulates the expression of CXCR2 on the surface of PMN remains unknown. Recent studies have suggested that NO down-regulates CXCR2 expression on neutrophils, which in turn leads to a reduction of the chemotactic response to the CXCR2-active ligand [30 ]. One interesting possibility is that H2S may function together with NO to regulate the expression of CXCR2. In vascular tissue, a number of potential cross-talk points between NO and H2S have been reported. For example, H2S may decrease the sensitivity of the cGMP pathway to NO and the expression level of NO synthase [6 ]. Clearly, further studies are necessary to dissect the mechanism underlying H2S-mediated CXCR2 regulation.

Although our findings suggest that H2S facilitates the leukocyte–endothelium interaction in sepsis, there are some earlier reports indicating that H2S may down-regulate leukocyte recruitment in inflammation [14 , 15 ]. The inconsistency between the present study and earlier study may be a result of the dose of H2S donor used. In the present study, we used NaHS at a dose of 10 mg/kg, which increases plasma concentration of H2S significantly and causes obvious lung inflammation [23 ]. In the earlier studies, three different H2S donors were applied at doses that approximated the physiological concentration of H2S. These contradictory observations in the present and earlier studies may also be a result of different animal models used and different organs investigated. In addition, the inhibitor of CSE used in earlier studies is different from those used in the present study. However, although the data in the present study and earlier study are conflicting, they raise the possibility that H2S may act as an anti-inflammatory mediator to inhibit the leukocyte–endothelium interaction at a physiological concentration but may contribute to inflammation in an already-inflamed area when overproduced. A similar circumstance has been reported with respect to another gaseous mediator, NO, which is also regarded as a double-edged sword. Synthesized in appropriate amounts, it contributes to blood-pressure regulation, neuronal communication, and immune defense. Conversely, excessive and uncontrolled production of NO is detrimental to several inflammatory diseases such as septic shock, autoimmune diseases, and some forms of chronic inflammation [31 ].

In summary, our results show that H2S elevates tissue levels of adhesion molecules and provokes leukocyte–endothelium interaction in sepsis through a mechanism involving NF-{kappa}B activation. It also facilitates chemoattractant-directed migration of PMN by up-regulation of chemokine receptors. The present study may contribute to the understanding of the precise mechanism underlying the proinflammatory role of H2S in inflammatory diseases.


   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. The authors have no financial conflict of interest. We thank Ms. Mei Leng Shoon for her help with the animal experiments and Mr. Akhil Kumar Hegde Rama for reading the manuscript.

Received April 23, 2007; revised May 21, 2007; accepted June 4, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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