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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6270fjev1 20/12/2118    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zanardo, R. C. O. Articles by Wallace, J. L. Search for Related Content PubMed PubMed Citation Articles by Zanardo, R. C. O. Articles by Wallace, J. L.

Hydrogen sulfide is an endogenous modulator of leukocyte mediated inflammation

Renata C. O. Zanardo^*, Vincenzo Brancaleone, Eleonora Distrutti, Stefano Fiorucci, Giuseppe Cirino and John L. Wallace^*,1

^* Inflammation Research Network, University of Calgary, Calgary, Alberta, T2N 4N1, Canada;

Department of Experimental Pharmacology, University of Naples, Naples, Italy; and

Division of Gastroenterology and Hepatology, University of Perugia, Perugia, Italy

¹Correspondence: Department of Pharmacology and Therapeutics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, T2N 4N1, Canada. E-mail: wallacej{at}ucalgary.ca

SPECIFIC AIMS

Hydrogen sulfide (H₂S) is increasingly recognized as a physiologically important signaling molecule, possibly contributing to innate immunity. The aims of this study were to 1) determine whether H₂S modulates leukocyte adherence to the endothelium; 2) determine whether H₂S inhibits leukocyte infiltration; and 3) examine the contribution of H₂S to edema formation.

PRINCIPAL FINDINGS

1. H₂S modulates leukocyte adherence to the vascular endothelium
The effects of H₂S donors on leukocyte adherence to the vascular endothelium were examined in the rat using intravital microscopy. Leukocyte adherence within mesenteric venules was observed in response to intragastric administration of aspirin (50 mg/kg) or superfusion with f-Met-Leu-Phe (fMLP; 10 µM). Each agent induced a time-dependent increase in leukocyte adherence during the 60 min observation period (Fig. 1 ). Both H₂S donors (Na₂S and NaHS, given orally) inhibited aspirin-induced leukocyte adherence (ED₅₀ of 5.0 µmol/kg for Na₂S). H₂S donors (Na₂S and NaHS at 100 µmol/kg; Lawesson’s reagent at 3 µmol/kg) also suppressed fMLP-induced leukocyte adherence. Inhibition of leukocyte adherence by the H₂S donors was reversed by pretreatment with glibenclamide, an ATP-activated potassium channel (K_ATP) antagonist.

View larger version (21K): [in this window] [in a new window]   Figure 1. Hydrogen sulfide inhibits aspirin-induced leukocyte adherence in mesenteric venules via activation of KATP channels. Na2S dose-dependently suppressed leukocyte adherence induced by intragastric aspirin (50 mg/kg). Inhibition of aspirin-induced adherence by Na2S (100 µmol/kg) was abolished by pretreatment with glibenclamide (10 mg/kg), a KATP channel antagonist. *P < 0.05 vs. corresponding vehicle-treated group. P < 0.05 vs. corresponding group receiving aspirin alone. Each group consisted of at least 5 rats. Results are plotted as mean ±SE.

We next investigated the possibility that endogenous H₂S could modulate leukocyte adherence in the absence of any inflammatory stimulus. H₂S is synthesized primarily via two enzymes: cystathionine--lyase (CSE) and cystathionine-ßbeta;-synthetase (CSB). Both enzymes are expressed in the rat mesentery. Oral administration of a reversible inhibitor of CSE, ßbeta;-cyanoalanine, resulted in a time-dependent increase in leukocyte adherence to the vascular endothelium, reaching > 10-fold above basal levels after 60 min. This inhibitor also caused a marked decrease in leukocyte rolling velocity (to 50% of basal levels).

2. H₂S reduces leukocyte infiltration via K_ATP channel activation
Administration of carrageenan into a rat air pouch resulted in infiltration of substantial numbers (6x10⁷) of leukocytes over the next 6 h, most of which (87.0±1.1%) were neutrophils. Pretreatment with H₂S donors (NaHS, N-acetylcysteine, Lawesson’s reagent; i.p.) dose-dependently reduced the numbers of leukocytes infiltrating into the air pouch (ED₅₀ of 42.7, 29.9, and 1.3 µmol/kg, respectively, defining the response to dexamethasone as maximal). With the highest dose of each H₂S donor tested, the reduction in leukocyte infiltration was comparable to that achieved by an NSAID (diclofenac), a NOS inhibitor (L-NAME), or a corticosteroid (dexamethasone).

N-Acetylcysteine is a precursor for L-cysteine, the substrate from which H₂S is synthesized via CSE and CBS. The ability of N-acetylcysteine to reduce carrageenan-induced leukocyte infiltration was dependent on CSE activity. Prior treatment with an inhibitor of CSE (ßbeta;-cyanoalanine) reversed the inhibitory effects of N-acetylcysteine. Moreover, ßbeta;-cyanoalanine increased basal leukocyte infiltration and that induced by carrageenan, suggesting that endogenous H₂S synthesis down-regulates leukocyte infiltration. The anti-inflammatory effects of N-acetylcysteine were also reversed by glibenclamide, a K_ATP channel antagonist.

3. H₂S modulates edema formation via K_ATP channel activation
Injection of carrageenan into the hind footpads of rats resulted in a rapid and marked increase in paw volume as a consequence of edema formation (Fig. 2 , upper panel). The increase in paw volume could be significantly reduced by pretreatment with diclofenac, an NSAID. Pretreatment with NaHS or Na₂S similarly decreased carrageenan-induced paw edema (ED₅₀ of 28 and 35 µmol/kg, respectively), as did pinacidil, a K_ATP channel agonist. In contrast, suppression of endogenous H₂S synthesis, through administration of ßbeta;-cyanoalanine, resulted in a significantly greater paw swelling response to carrageenan. The reduction of carrageenan-induced paw edema by H₂S donors (NaHS or Na₂S) could be reversed by pretreatment with glibenclamide, a K_ATP channel antagonist (Fig. 2 , lower panel).

View larger version (22K): [in this window] [in a new window]   Figure 2. Hydrogen sulfide suppresses edema formation in the rat paw in a KATP channel-dependent manner. Top panel: injection of carrageenan into the rat hindpaw resulted in significant edema formation over the ensuing 5 h. Pretreatment with an H2S donor (NaHS; 100 µmol/kg i.p.) significantly reduced paw edema at each time point (P<0.05), as did pretreatment with a conventional nonsteroidal anti-inflammatory drug (diclofenac, 10 mg/kg i.p.) and a KATP channel agonist (pinacidil; 10 mg/kg i.p.). In contrast, administration of an inhibitor of cystathionine -lyase (ßbeta;-cyanoalanine; 10 mg/kg i.p.) significantly increased the edema formation induced by carrageenan at all time points (P < 0.05). Bottom: the increase in paw edema occurring during the first hour after carrageenan administration is shown. The reduction of paw edema by either of two hydrogen sulfide donors (NaHS and Na2S, each at 100 µmol/kg) was abolished by pretreatment with glibenclamide, a KATP channel antagonist (10 mg/kg i.p. 30 min before carrageenan); glibenclamide alone did not alter carrageenan-induced edema formation. Data are shown as mean ± SE, with at least 5 rats per group.

CONCLUSIONS AND SIGNIFICANCE

H₂S is an important modulator of vascular tone and acts as a neuromodulator. The present study demonstrates that H₂S also plays important roles in inflammation (Fig. 3 ). Several H₂S donors were shown to suppress leukocyte adherence to the vascular endothelium and to reduce leukocyte infiltration and edema formation. The effects of H₂S were seen irrespective of the inflammatory stimulus used (carrageenan, aspirin, fMLP). Suppression of endogenous H₂S synthesis through blockade of CSE resulted in enhanced leukocyte adhesion, leukocyte infiltration, and edema formation. These actions appeared to be mediated via K_ATP channels, as they were reversed by glibenclamide and mimicked by pinacidil. Our observations therefore suggest an important role for endogenous H₂S as a modulator of several key components of acute inflammatory responses, particularly those occurring at the leukocyte-endothelial interface (Fig. 3) .

View larger version (24K): [in this window] [in a new window]   Figure 3. Hydrogen sulfide modulates inflammatory processes at the leukocyte-endothelial interface. A) H2S is synthesized in blood vessels via cystathionine--lyase (CSE), which is expressed in endothelial cells and smooth muscle cells. H2S tonically down-regulates leukocyte adherence via activation of ATP-activated potassium channels (KATP) on leukocytes and endothelium. B) When endogenous H2S synthesis is inhibited, such as with ßbeta;-cyanoalanine, leukocyte rolling and adherence to the vascular endothelium increase, likely due partly to elevated expression of adhesion molecules on leukocytes (CD11/CD18) and endothelial cells (P-selectin). Marked increases in endothelial permeability that result in edema formation occur when H2S synthesis is suppressed.

As for other gaseous mediators (carbon monoxide, NO), H₂S was recognized for its toxicity long before its importance in physiological processes was described. H₂S is synthesized, primarily from L-cysteine, through the actions of the enzymes CSE and CBS. In rats, blood and plasma levels of H₂S are in the 10–100 µM range. In the present study, we used three different H₂S donors at doses that would approximate concentrations of H₂S within the physiological range. The observation that suppression of endogenous H₂S synthesis with ßbeta;-cyanoalanine led to increased leukocyte adherence and infiltration is consistent with a role for this mediator as a tonic inhibitor of leukocyte adherence/extravasation.

Of the four H₂S donors used in this study, only N-acetylcysteine requires metabolism for H₂S to be released. N-acetylcysteine is a precursor of L-cysteine, which is the substrate for H₂S generation via CSE and/or CBS. The observation that the anti-inflammatory actions of N-acetylcysteine were reversed by an inhibitor of CSE (ßbeta;-cyanoalanine) is consistent with the effects being mediated by H₂S.

NO is another gaseous mediator that exerts many effects in common with H₂S in the cardiovascular and nervous systems. Moreover, there is evidence of cross-talk between H₂S and NO on many levels. For example, H₂S promotes the release of NO from vascular endothelium, whereas an NO donor was shown to increase the conversion of L-cysteine to H₂S at least in part by increasing the expression of CSE, a key enzyme for H₂S synthesis. Hemoglobin (Hb) has been referred to as a common "sink" for H₂S, NO, and carbon monoxide. Thus, saturation of Hb binding to one of these gaseous mediators could lead to enhanced plasma levels and to biological effects from the others.

While our findings point to a role for H₂S as an endogenous modulator of inflammation, there are reports suggesting that this mediator may contribute to inflammatory processes. In addition to reports that irreversible inhibition of CSE can attenuate the severity of experimental pancreatitis and endotoxemia, administration of DL-propargylglycine has been shown to dose-dependently reduce carrageenan-induced paw edema. It is possible that with a very high level of suppression of H₂S synthesis, a significant decrease in blood flow would be anticipated that would result in reduced edema formation.

In summary, the results of this study have demonstrated a role for endogenous H₂S as a modulator of key inflammatory events occurring at the interface of leukocytes and the vascular endothelium. The data suggest that H₂S is a tonic regulator of leukocyte adherence to the endothelium and of endothelial permeability. The anti-inflammatory effects of H₂S appear to be mediated through K_ATP channels. These results therefore identify H₂S, the key enzyme responsible for H₂S synthesis, and K_ATP channels as potential targets for novel anti-inflammatory therapies.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6270fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6270fjev1 20/12/2118    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zanardo, R. C. O. Articles by Wallace, J. L. Search for Related Content PubMed PubMed Citation Articles by Zanardo, R. C. O. Articles by Wallace, J. L.