HYDROGEN SULFIDE IMPROVES NEUTROPHIL MIGRATION AND SURVIVAL IN

SEPSIS VIA K+

ATP CHANNEL ACTIVATION

Fernando Spiller1, Maria I.L. Orrico

1, Daniele C. Nascimento

1, Paula G. Czaikoski

1, Fabrício O.

Souto3, José C. Alves-Filho

1, Andressa Freitas

1, Daniela Carlos

1, Marcelo F. Montenegro

1,

Alberto F. Neto4, Sergio H. Ferreira

1, Marcos A. Rossi

2, John S. Hothersall

1, Jamil Assreuy

5 and

Fernando Q. Cunha1.

Departments of 1Pharmacology,

2Pathology, and

3Surgery and Anatomy, School of Medicine of

Ribeirao Preto; 4Department of Pharmaceutical Science, Faculty of Pharmaceutical Sciences,

University of Sao Paulo, Ribeirao Preto, Sao Paulo and 5Department of Pharmacology, Federal

University of Santa Catarina, Florianopolis, SC, Brazil.

Corresponding author: Prof. Dr. Fernando Q. Cunha, Department of Pharmacology, Faculty of Medicine of Ribeirao

Preto, University of Sao Paulo, Avenida Bandeirantes, 3900, 14049-900, Ribeirao Preto, Sao

Paulo, Brazil. Tel. 55 16 3602 3324, Fax. 55 16 3633 2301. E-mail: fdqcunha@fmrp.usp.br

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São

Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),

Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq) and Programa de

Núcleos de Excelência (PRONEX).

Running Head: H2S improves survival in sepsis

Subject descriptor number: 4.12

Word count: 3764

This article has an online data supplement, which is accessible from this issue's table of contents

online at www.atsjournals.org.

Scientific Knowledge on the Subject

During the inflammatory process, the cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S)

pathway has been shown to be involved in the control of leukocyte infiltration into inflammatory

sites. However, the role of this pathway on the regulation of leukocyte recruitment to infection

sites during sepsis has been not investigated.

What This Study Adds to the Field

H2S restores neutrophil migration to the infectious focus and improves survival outcome in

severe sepsis by a K+

ATP channel-dependent mechanism.

Page 1 of 46 Media embargo until 2 weeks after above posting date; see thoracic.org/go/embargo

AJRCCM Articles in Press. Published on March 25, 2010 as doi:10.1164/rccm.200907-1145OC

Copyright (C) 2010 by the American Thoracic Society.

1

ABSTRACT

Rationale: Recovering the neutrophil migration to the infectious focus improves survival in

severe sepsis. Recently, we demonstrated that the cystathionine γ-lyase (CSE)/hydrogen sulfide

(H2S) pathway increased neutrophil recruitment to inflammatory focus during sterile

inflammation. Objectives and methods: Evaluate if H2S administration increases neutrophil

migration to infectious focus and survival of mice with sepsis induced by cecal ligation and

puncture (CLP). Results: The pre-treatments of mice with H2S donors (NaHS or Lawesson’s

reagent) improved leukocyte rolling/adhesion in the mesenteric microcirculation as well as

neutrophil migration. Consequently, bacteremia levels were reduced, hypotension and lung

lesions were prevented and the survival rate was enhanced from ~13% to ~80%. Notably, even

when treatment was delayed (6 h post-CLP), a highly significant reduction in mortality compared

to untreated mice was still observed. Moreover, H2S-pretreatment prevented the down-regulation

of CXCR2 and L-selectin and the up-regulation of CD11b and GRK2 in neutrophils during

sepsis. H2S also prevented the reduction of ICAM-1 expression in the endothelium of the

mesenteric microcirculation in severe sepsis. Confirming the critical role of H2S on sepsis

outcome, pretreatment with dl-propargylglycine (PAG, a CSE inhibitor) inhibited neutrophil

migration to the infectious focus, enhanced lung lesions and induced high mortality in mice

subjected to non-severe sepsis (from 0% to ~80%). Finally, the beneficial effects of H2S were

blocked by glibenclamide (a K+

ATP channel blocker). Conclusion: These results showed that H2S

restores neutrophil migration to the infectious focus and improves survival outcome in severe

sepsis by a K+

ATP channel-dependent mechanism.

Word count: 244

Keywords: neutrophil migration failure; sepsis; hydrogen sulfide; survival; cecal ligation and

puncture.

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INTRODUCTION

Sepsis is one of the major causes of death in intensive care units and has a mortality rate of

30 to 50%. Therapy for severe sepsis and septic shock is still largely symptomatic and supportive

(1).

We have shown that severe sepsis induced by cecal ligation and puncture (CLP) (2) or by

Gram-positive (3) or Gram-negative (4) intraperitoneal (ip) bacterial inoculation is associated

with inhibition of neutrophil recruitment to the sites of infection, bacteremia and high mortality

rates. Similarly, neutrophils isolated from non-surviving septic patients exhibited a suppressed

chemotactic response when compared to neutrophils from surviving septic patients or healthy

volunteers (5, 6). In investigating the mechanisms involved in the reduction of neutrophil

migration in severe experimental sepsis, we have shown that circulating cytokines and

chemokines and/or systemic TLR2/4 activation reduce the rolling, adhesion and chemotaxis of

neutrophils (7). The reduction of these events either in experimental or septic patients is due to

CXCR2 internalization and modulation of the levels of adhesion molecules on neutrophils (8-

10). The internalization of CXCR2 is associated with up-regulation of G protein-coupled

receptor kinase 2 (GRK2) expression in neutrophils (8, 11).

Hydrogen sulfide (H2S) is synthesized from L-cysteine mainly by cystathionine β-synthase

(CBS) and cystathionine γ-lyase (CSE) (12). The CSE/H2S pathway has been demonstrated to

play important roles in different biological systems. It modulates blood pressure (13) and

leukocyte trafficking in several inflammatory models such as acute pancreatitis (14), hindpaw

edema (15) and endotoxemia (16). In experimental sepsis induced by CLP, the CSE/H2S

pathway has also been shown to be involved in the control of leukocyte infiltration into distant

tissues such as lung and liver (17, 18).

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The role of the CSE/H2S pathway in regulating leukocyte recruitment to inflammatory sites

is not completely understood and sometimes contradictory. It has been shown that H2S donors

(NaHS and Na2S) inhibited aspirin-induced leukocyte adherence in mesenteric venules (19, 20).

However, Zhang et al. (17) have shown that the pretreatment of mice with dl-propargylglycine

(PAG), the CSE inhibitor, inhibited CLP-induced leukocyte-endothelial interactions in

mesenteric venules, whereas NaHS improved these parameters. In agreement with this, we have

shown that treatment of mice with H2S synthesis inhibitors reduced neutrophil migration into the

peritoneal cavities or femur/tibial joints of mice. Predictably, treatment of animals with H2S

donors enhanced these parameters (11). Therefore, our objective was to evaluate if exogenous

H2S increases neutrophil migration to the infectious focus and the survival of mice with sepsis

induced by CLP. Moreover, we also investigated whether the mechanism of action of H2S on

neutrophil migration was mediated via the ATP-dependent K+ (K

+ATP) channel pathway because

many of the other effects associated with H2S have been attributed to K+

ATP channel activation

(12).

MATERIALS AND METHODS

Experimental details are provided in the online supplement.

Word count: 500

Animals

The research was approved by the Animal Research Ethics Committee of the FMRP. Male

Swiss mice (23–26 g) were used.

Sepsis model

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Sepsis was induced by a CLP model, as described (21). We have standardized 2 punctures

in the cecum with a 26-gauge needle as non-severe (NS) sepsis induction or 2 punctures with an

18-gauge needle as severe (S) sepsis induction.

Neutrophil migration to the peritoneal cavity

Neutrophil migration was assessed 6 h after CLP, as reported (2).

Bacterial counts

The bacterial count was determined in the blood 6 h after CLP, as described (4).

Intravital microscopy

Leukocyte rolling and adhesion were examined in the mesenteric microcirculation by

intravital microscopy 2 h and 4 h after CLP, respectively, as described (22).

Cystathionine Gamma Lyase Assay

Total leukocytes were purified 2 h after CLP, and CSE activity measured as described (23).

Determination of Cytokine Levels in the Peritoneal Exudates and Blood

Animals were sacrificed 6 h after CLP, and the peritoneal lavage and blood were collected.

Cytokine concentrations were determined by ELISA.

Histological examination

The animals were sacrificed 12 h after CLP, and after isolation the lungs were fixed by

immersion in 5% paraformaldehyde, dehydrated and embedded in paraffin wax. Sections of 3-

µm thickness were stained with hematoxylin and eosin for histological examination. A

pathologist performed blind histological assessment.

Isolation of bone marrow neutrophils

Bone marrow cells were collected, and neutrophils were isolated by differential

centrifugation on a Percoll gradient, as described (8).

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Chemotaxis Assays

Mouse bone marrow neutrophils were incubated in the absence (control) or in the presence

of glibenclamide (GLB, a K+

ATP channel blocker) or the H2S donor sodium hydrogen sulfite

(NaHS, 30, 100 or 300 µM). In addition, one group was incubated (37°C) with GLB (100 µM,

30 min) and then NaHS (300 µM) for 1 h. Following this, neutrophil chemotaxis was stimulated

by MIP-2 in a Boyden chamber.

Flow Cytometry Analysis of CD11b, CD62L and CXCR2 expression

Flow cytometry was analyzed with a flow cytometer (BD FACSort; Mountain View, CA)

according to a previously described procedure (9). Total blood was collected 6 h after CLP and

stained with labeled antibodies: PE-anti-CXCR2 (R&D Systems), PE-anti-CD62L, FITC-CD11b

and PerCP-anti-Gr-1 (BD Biosciences).

Immunofluorescence assay for ICAM-1/CD54 or G protein-coupled receptor kinase 2

(GRK2)

Six hours after CLP, an ICAM-1/CD54 immunofluorescence assay was performed in

mesenteric and lung tissues or GRK2 in neutrophils according to a previously described method

(11).

Statistical analysis

The data were reported as the mean ± SEM of values obtained from two or three

independent experiments (n = 5 in each experiment; exception for survival analyses, n = 10-12).

The means between different groups were compared by analysis of variance (ANOVA). If

significance was determined, individual comparisons were subsequently tested with Bonferroni's

t test for unpaired values. Moreover, the Kolmogorov-Smirnov test was employed to test for

normality. Because the rolling and adhesion results failed to reach normality, the data were log

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transformed to reach this assumption, and then ANOVAs were performed with the modified

data.

RESULTS

Hydrogen sulfide protects against CLP-induced lethality

Firstly, we evaluated CSE activity in blood leukocyte extracts 2 hours after sepsis induction

by CLP. Fig. 1A shows that in mice subjected to severe (S) sepsis, there was significantly

increased CSE activity when compared to sham-operated (Sham) mice, and PAG (1 mM)

abrogated this activity.

Following this, the effect of H2S-synthesis inhibition on survival during NS sepsis (animals

experiencing local infection) was determined (Fig. 1B). Mice pretreated with PAG (50 mg/kg,

ip) and subjected to NS sepsis exhibited a significant reduction (~80%) in survival rate when

compared with NS animals pretreated with saline. Moreover, PAG treatment in mice subjected to

moderate sepsis (~50% mortality rate) also increased the mortality rate (~100%, data not shown).

Furthermore, animals submitted to S sepsis and pretreated with saline or PAG failed to survive

beyond the second day after CLP. All Sham mice survived for 8 days after CLP (data not

shown).

In addition, PAG pretreatment inhibited neutrophil migration to the focus of infection (Fig.

2A), increased blood bacterial counts (Fig. 2B) and the levels of serum TNF-α, MIP-2 and KC

(Table E1) in NS septic mice. As shown in Fig. 2A, pretreatment of S septic mice with PAG did

not significantly change the number of neutrophils in the peritoneal cavity.

The effects of exogenous H2S on the survival rate of mice subjected to S sepsis were then

investigated. The sc pretreatment of mice subjected to S sepsis with 100 µmol/kg of NaHS (Fig.

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1C) or Lawesson’s reagent (Fig. 1E) improved the survival rate from 10-15% to ~80% 8 days

after CLP. The therapeutic treatment of S CLP mice with these H2S donors 6, 12, and 24 hours

post S sepsis induced a delay in mortality and a significant increase in the survival rate (~35%;

Fig. 1D and 1F).

As previously demonstrated (6), mice subjected to S sepsis showed a significantly reduced

neutrophil migration compared to the NS group (Fig. 2A). The sc pretreatment of mice subjected

to S sepsis with 100 µmol/kg of H2S donors prevented the failure of neutrophil migration to the

focus of infection (Fig. 2A), reduced blood bacterial count (Fig. 2B) and prevented hypotension

(Fig. E2B).

The sc administration of NaHS at a dose of 100 µmol/kg in naive mice did not induce

significant changes in mean arterial pressure (MAP, Fig. E2A). However, in accordance with

previous descriptions (24), the intravenous bolus injection of the same NaHS dose either in naive

(Fig. E2A) or septic mice (data not shown) provoked a significant decline in MAP compared to

saline injected mice.

Using a sulfide-sensitive electrode, we confirmed that plasma H2S levels were increased

after experimentally induced severe sepsis (Fig. E1A). Moreover, NaHS and Lawesson’s reagent

were able to release H2S in a concentration-dependent manner in vitro (data not shown). In

addition, the plasma levels of H2S increased significantly in mice sc treated with 1000 µmol/kg

NaHS (Fig. E1B).

Leukocyte rolling/adhesion to mesenteric microcirculation is improved by NaHS

PAG pretreatment of NS septic mice increased TNF-α and MIP-2 levels in peritoneal

exudates when compared to Sham or NS septic mice (Fig. E3A and E3B, respectively). The

levels of KC were significantly increased in S, NS and NS-PAG-pretreated septic mice when

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compared to Sham mice (Fig. E3C). Treatment with NaHS did not change the levels of these

cytokines in the peritoneal cavities of S septic mice (Fig. E3). These data strongly suggested that

the reduction of neutrophil migration to the site of infection after PAG treatment or in S sepsis

was not a consequence of low concentrations of cytokines and chemokines in the peritoneal

cavity. However, treatment with PAG inhibited the significant increase in rolling and adhesion of

leukocyte to endothelial cells of mesenteric venules in NS septic mice (Fig. 2C). This reduction

in leukocyte rolling and adhesion was also observed in S septic mice. Interestingly, when S mice

were pretreated with NaHS, there was a significant improvement in leukocyte rolling and

adhesion (Fig. 2C).

Hydrogen sulfide prevents acute lung injury in sepsis

Sepsis led to significant changes in pulmonary histology consistent with acute lung injury.

This included septal thickening, edema and congestion, hemorrhage and both neutrophil and

mononuclear cell accumulation. Animals submitted to S sepsis (Fig. E4E) showed more

pronounced changes in the lungs compared with the lungs of mice submitted to NS septic injury

(Fig. E4C). Following PAG treatment, these same groups presented significant aggravation of

lung alterations (Fig. E4F and E4D). Pretreatment of mice submitted to S sepsis with NaHS

significantly abrogated the development of acute lung injury (Fig. E4B), and the lung structure of

these mice appeared similar to the normal structure observed in Sham mice (Fig. E4A), except

for discrete interstitial neutrophil and mononuclear cell infiltration and edema.

KATP channel blocking prevents protective effects of H2S during sepsis

We evaluated if H2S improves neutrophil migration in a KATP channel-dependent fashion

using both in vivo and in vitro models. Bone marrow neutrophils incubated for 1 h (37°C) with

NaHS (300 µM) showed a significant increase (P < 0.001) in MIP-2-induced neutrophil

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chemotaxis when compared to the RPMI control group (Fig. 3A). When neutrophils were pre-

incubated with glibenclamide (GLB 100 µM, a selective KATP channel blocker), the MIP-2-

induced chemotaxis and the NaHS enhancement of MIP-2-induced chemotaxis were blocked

(Fig. 3A).

In vivo, pretreatment with GLB (40 µmol/kg, sc) inhibited neutrophil migration (Fig. 3B)

and increased mortality (Fig. 3C) of mice subjected to NS sepsis. Moreover, GLB treatment prior

to NaHS or Lawesson’s reagent treatment inhibited the improvement of neutrophil migration

(Fig. 3B) and survival (Fig. 3C) evoked by these H2S donors in S septic mice.

Because CD11b, CD62L and CXCR2 expression in leukocytes is necessary for neutrophil

rolling, adhesion and migration to the focus of infection (9), the expression of these receptors on

blood neutrophils was evaluated 6 h after CLP. S septic mice had significantly (P < 0.05)

decreased CD62L (Fig. 3E) and CXCR2 (Fig. 3F) expression, while CD11b (Fig. 3D) expression

increased in neutrophils when compared to neutrophils from NS or Sham mice. In contrast,

pretreatment of S septic animals with Lawesson’s reagent partially inhibited the down-regulation

of CD62L and CXCR2 and the up-regulation of CD11b in neutrophils. These effects of the H2S

donor were prevented by GLB pretreatment. Furthermore, when NS mice were pretreated with

PAG or GLB, the neutrophil expression of CD62L (Fig. E5B) and CXCR2 (Fig. E5C) was

significantly lower when compared to neutrophils from NS mice. PAG pretreatment of NS mice

also significantly raised the expression of CD11b, whereas GLB had no significant effect (Fig.

E5A).

High expression of GRK2 is involved in the internalization of chemokine receptors in

leukocytes (8). Six hours after severe sepsis induction, we observed a significant augmentation

of GRK2 expression in blood neutrophils (Fig. 4E). Pretreatment of NS mice with PAG (Fig. 4C)

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or GLB (Fig. 4D) enhanced, whereas Lawesson’s reagent (Fig. 4F) pretreatment reduced, S

sepsis-induced GRK2 expression. Moreover, GLB prevented the inhibitory effect of H2S on

GRK2 expression (Fig. 4G). These results suggest that H2S prevents CXCR2 internalization via

inhibition of GRK2 expression by a KATP channel-dependent mechanism.

H2S prevented severe sepsis-induced ICAM-1 down-modulation by KATP channel activation

To further clarify the mechanisms by which H2S increased neutrophil recruitment to an

infectious focus, we evaluated ICAM-1 levels in the endothelium of mesenteric microcirculation

after sepsis induction. Compared with Sham animals (Fig. 5A), NS sepsis (Fig. 5B) induction

increased ICAM-1 expression in mesenteric vessels, whereas pretreatment of these mice with

PAG (Fig. 5C) or GLB (Fig. 5D) or S sepsis induction (Fig. 5E) inhibited ICAM-1 expression.

Pretreatment of S septic mice with NaHS significantly enhanced ICAM-1 expression in

endothelial cells (Fig. 5F), and GLB blocked this increase in ICAM-1 expression (Fig. 5G).

We also evaluated the levels of ICAM-1 expression in lung vessels after sepsis induction.

Similar to mesenteric vessels, NS sepsis induced a significantly rise in ICAM-1 expression in

lung vessels, which was inhibited by PAG pretreatment. In contrast, S sepsis reduced ICAM-1

expression and pretreatment with NaHS resulted in a significant increase in ICAM-1 expression

levels (Fig. E6). These data are in accordance with previous reports showing that there is an up-

regulation of ICAM-1 in the lungs during sepsis and that PAG treatment is able to prevent this

increase, while H2S donors increase the ICAM-1 expression (17).

DISCUSSION

In the present study, we demonstrate that pretreatment of mice submitted to severe sepsis

with H2S donors prevented the failure of neutrophil migration, reduced bacterial spreading,

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prevented hypotension and improved the survival of the mice from ~12% to ~80%. Furthermore,

and more clinically relevant, the delayed treatment of mice with these donors improved the

survival rate by up to ~35%.

The CSE H2S-forming enzyme activity and mRNA levels have been reported as increased

in the liver and kidneys after endotoxin- or CLP-induced sepsis (16, 18). However, the role of

H2S in systemic inflammatory response syndrome (SIRS) is not understood. There is evidence

that the inhibition of H2S synthesis by PAG decreases leukocyte infiltration in the liver and/or

lungs after acute pancreatitis or endotoxemia induction (16, 25). Moreover, Zhang et al. (18)

have shown that administration of PAG at the same dose and route as those used in the present

study (50 mg/kg, ip) significantly reduced leukocyte accumulation in the lungs and livers of

CLP-induced septic animals and attenuated the mortality of animals. Subsequently, the same

group showed that treatment of CLP-septic mice with PAG inhibited rolling and adhesion of

leukocytes to the mesenteric microcirculation (17). Rolling and adhesion are critical steps for

neutrophil migration from the bloodstream into tissues during the inflammatory process (26).

Therefore, it is reasonable to conclude that the reduction of rolling and adhesion of leukocytes in

the mesenteric microcirculation also reduces neutrophil migration into the peritoneal cavity in

CLP-induced sepsis (focus of the infection). In fact, herein we showed that the significant

reduction of leukocyte rolling and adhesion on the mesenteric microcirculation induced by

inhibiting H2S biosynthesis resulted in the reduction of neutrophil migration to the focus of

infection. Therefore, the decrease in available H2S allowed bacterial spread, increased systemic

inflammatory response (measured by cytokines TNF-α, MIP-2, KC in serum), and contrary to

the findings of Zhang’s group (23), aggravated lung injury and induced higher mortality. The

data emphasize the importance of CSE activity and consequent H2S production in infection

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control and host survival. Thus, the rise of H2S production observed in severe sepsis might be a

protective mechanism triggered by the host aimed at enhancing neutrophil migration to the focus

of infection in an attempt to confine and combat the infection. Similarly, we observed a

significant increase in CSE activity in blood leukocytes and plasma H2S levels after severe sepsis

induction. However, it would appear that the amount of H2S generated in this condition is not

sufficient to protect against mortality. Thus, when we administered pharmacological

concentrations of H2S to S septic animals, they presented 80% survival, reinforcing our

hypothesis that the level of endogenously produced H2S was insufficient in this

pathophysiological condition. Experimental differences, including health status, age and gender

of the animals, commercial source of PAG and level of severity of sepsis, may explain the

opposing effects of CSE inhibition on survival and lung injury of mice exposed to CLP-induced

sepsis, being the deleterious effects described in the present study and the protective effects

described by Zhang´s group (18).

Typically, migration of neutrophils to a site of infection involves rolling on activated

endothelium (mediated by selectins) and subsequently firm adherence to the endothelium via

interaction of activated neutrophil integrins with the endothelial cell intercellular adhesion

molecules (ICAMs) (27). Here, we observed that mice subjected to NS sepsis, which had

efficient rolling/adhesion and neutrophil migration, expressed CD62L, CD11b and ICAM-1 at

similar levels to that found in Sham mice, whereas mice with S sepsis, which presented failure of

neutrophil migration, had a significant shedding of CD62L, high expression of CD11bin

circulating neutrophils and low expression of ICAM-1 in the mesenteric endothelium (28, 29).

Treatment with H2S donors prevented L-selectin shedding, down-modulation of ICAM-1 and the

increase of CD11b during S sepsis. Consequently, mice treated with H2S donors presented

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leukocyte rolling and adherence on the endothelium similar to that observed in mice with NS

sepsis.

The microbicidal activity of the migrating cells is fundamental to the containment of

infection (30). In view of this, we observed that both in vivo and in vitro treatments with PAG or

H2S donors did not change neutrophil phagocytic or killing capacities. Moreover, H2S did not

show antimicrobial activity as a single agent (data not shown). These results reinforce that the

beneficial effects of H2S on S sepsis are mainly the result of the reestablishment of neutrophil

migration to the focus of infection.

As discussed above, the inability of the host to restrict the infection locally triggers a

systemic inflammatory process, characterized, among other events, by the accumulation of

leukocytes into peripheral organs including the lungs, which contributes to tissue lesion and

overall organ dysfunction (31). Accordingly, treatments of the animals with an H2S donor or

PAG reduced and increased the lung lesion, respectively. Interestingly, H2S treatment of severe

septic mice was able to increase ICAM-1 expression in lung vessels. However, in this group,

there was a dramatic reduction in leukocyte infiltration. The direct explanation for this apparent

contradiction is that H2S treatment also reduced the increase of CD11b expression in the

neutrophils; which is necessary to trigger neutrophil accumulation in the lung and other organs

during systemic inflammation (32).

The aggravation of sepsis syndrome correlates with progressive hypotension that is

unresponsive to vasoconstrictors (33). Several studies have reported an important role of H2S in

the cardiovascular system (12). Genetic deletion of CSE increased blood pressure, whereas

intravenous bolus injection of H2S reduced blood pressure dose-dependently (2.8-39 µmol/Kg)

(13, 24). In our experiments, we also observed that intravenous bolus administration of NaHS

decreased blood pressure. On the other hand, subcutaneous administration of NaHS did not

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reduce the mean arterial pressure of naïve mice; however, it prevented CLP-induced

hypotension. This valuable effect of H2S on hypotension is unlikely to be due to a direct effect

on the vasculature. Probably, it is a consequence of the reestablishment of infection control

evoked by H2S in the septic animals. Alternatively, it is possible that there is an interaction of

H2S with the NO/iNOS system, because during endotoxic shock H2S was able to prevent the

expression of iNOS and, consequently, LPS-induced hypotension (34).

The CXC-ELR+ chemokines and their receptors are key factors for host defense against

infection (35, 36). In this context, CXCR2 antagonists, which block neutrophil influx, are shown

to increase septic lethality (9). Interestingly, during S sepsis, the down-regulation of CXCR2 in

blood neutrophils was associated with reduction of adhesion and chemotaxis of leukocytes (8, 9,

37). GRK2 has been implicated in the down-regulation of chemokine receptors (CXCRs and

CCRs) (8, 11, 38). Here, we have shown that the treatment of mice subjected to S sepsis with

H2S donors was able to prevent up-regulation of GRK2 expression and down-regulation of

CXCR2 in blood neutrophils. These data are in accordance with previous reports that CXCR2 is

up-regulated in blood neutrophils from naïve mice after NaHS treatment (17).

Studies have reported that several pharmacological effects of H2S are related to the opening

of KATP channels (12). In the present work, we have shown that the H2S-stimulated improvement

in survival during sepsis is mediated by KATP channel activation. KATP channel activity has

already been shown to be important to neutrophil migration. In fact, treatments of animals with

inhibitors or openers of KATP channels inhibit and enhance neutrophil migration in different

experimental models, respectively (39). In the present study, it was observed that GLB treatment

mimicked the action of PAG and prevented the effects of H2S on neutrophil migration, CXCR2

internalization and GRK2 or ICAM-1 expression. Taken together, our results suggest that the

effect of H2S in sepsis is mediated by KATP channel activation. In this context, Tang et al. (40),

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using a whole-cell and single-channel patch-clamp technique, demonstrated that exogenous H2S

activated KATP channels and hyperpolarized cell membranes in rat mesenteric artery vascular

smooth muscle cells. Inhibition of endogenous H2S production with PAG also reduced whole-

cell KATP currents. Moreover, H2S modifies a large number of proteins, including actin and the

Kir6.1 subunit of the KATP channel, by S-Sulfhydration, a process that may regulate their

function (41).

In summary, our data show a critical role of endogenous H2S production in efficient

neutrophil migration to the infectious focus in NS sepsis. Additionally, exogenous H2S prevents

the failure of neutrophil migration to the focus of infection, and both pre- and post-treatment

ultimately improved the survival in S sepsis. In this context, H2S post-treatment has also been

shown to significantly decrease mortality in a murine model of acute lung injury (42). Therefore,

H2S donors could be important agents in a new approach toward sepsis treatment.

Acknowledgments

We thank GB Francisco for technical assistance.

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19. Fiorucci S, Antonelli E, Distrutti E, Rizzo G, Mencarelli A, Orlandi S, Zanardo R, Renga

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39. Da Silva-Santos JE, Santos-Silva MC, Cunha Fde Q, Assreuy J. The role of atp-sensitive

potassium channels in neutrophil migration and plasma exudation. J Pharmacol Exp Ther

2002;300:946-951.

40. Tang G, Wu L, Liang W, Wang R. Direct stimulation of k(atp) channels by exogenous

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41. Mustafa AK, Gadalla MM, Sen N, Kim S, Mu W, Gazi SK, Barrow RK, Yang G, Wang

R, Snyder SH. H2s signals through protein s-sulfhydration. Sci Signal 2009;2:ra72.

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effect of hydrogen sulfide in a murine model of acute lung injury induced by combined burn and

smoke inhalation. Clin Sci (Lond) 2008;115:91-97.

FIGURES LEGENDS

Figure 1. Survival rate deterioration with CSE inhibitor dl-propargylglycine (PAG) and

improvement with hydrogen sulfide donors (NaHS and Lawesson’s reagent) in mice

subjected to sepsis. (A) CSE activity was evaluated in blood leukocytes 2 h after surgery in

mice subjected to sham-operation (Sham), non-severe (NS) or severe (S) sepsis by cecal ligation

and puncture (CLP). CSE activity was also evaluated in blood leukocytes from S septic mice in

the presence of PAG (1 mM). *P < 0.01 compared to the Sham group and #P < 0.01 compared to

the S group (ANOVA, followed by Bonferroni’s test). One hour before induction of Sham, NS or

S sepsis, mice were given saline (Sal), (B) PAG (50 mg/kg, ip), (C) NaHS (10, 30 or 100

µmoles/kg, sc) or (E) Lawesson’s reagent (100 µmoles/kg, sc). Six h, twelve h and twenty-four h

after S sepsis induction, previously untreated mice were randomly given (D) NaHS (100

µmol/kg, sc), (F) Lawesson’s reagent (100 µmoles/kg, sc) or Sal (sc). Their survival rates were

monitored over 8 days. The results are expressed as percent of survival. *P < 0.05 compared to

NS plus Sal, and **P < 0.05 compared to S plus Sal. Mantel-Cox log-rank test (n = 12–20).

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Figure 2. The H2S donors (NaHS and Lawesson’s reagent) prevented the failure of

neutrophil migration to the infectious focus, while PAG inhibited neutrophil migration in

mice subjected to sepsis. One hour before induction of sham-operation (Sham), non-severe (NS)

or severe (S) sepsis, mice were given PAG (10, 50 or 100 mg/kg, ip), NaHS (10, 30 or 100

µmoles/kg, sc), Lawesson’s reagent (10, 30 or 100 µmoles/kg, sc) or saline (Sal). (A) Six hours

after surgery, neutrophil migration into the peritoneal cavity was determined. The data are

reported as the mean ± SEM x 106 neutrophils/cavity. *P < 0.001 compared to the NS plus Sal

group and #P < 0.001 compared to the S plus Sal group. (ANOVA, followed by Bonferroni’s

test, n = 5). (B) Bacterial counts in the blood of mice pretreated with PAG (50 mg/kg, ip), NaHS

(100 µmol/kg, sc) or Sal were determined 6 h after CLP. The data are reported as the mean of the

Log of CFU per 10 µL. *P < 0.01 compared to NS plus Sal; #P < 0.01 compared to the group S

plus Sal (t test, followed by Mann-Whitney U test, n = 5). (C) Animals were treated with Sal,

PAG (50 mg/kg, ip) or NaHS (100 µmol/kg, sc) 1 hour before Sham, NS or S sepsis induction.

Rolling and adherent leukocytes in the mesenteric microcirculation were evaluated 2 h and 4 h

after surgery, respectively. The data are reported as the mean ± SEM of leukocyte rolling per min

and adherent cells per 100 µm2 of vessel. *P < 0.05 compared to Sham; **P < 0.05 compared to

NS plus Sal; and #P < 0.05 compared to S plus Sal (ANOVA, followed by Bonferroni’s test, n =

5). ND = not detectable.

Figure 3. KATP channel blockage prevented the H2S effects in vivo and in vitro. (A) Isolated

neutrophils were incubated (1 h) with H2S, (NaHS, 300 µM), glibenclamide (GLB 100 µM) or

GLB (100 µM) plus H2S (NaHS, 300 µM) for another 30 min before measuring chemotaxis

induced by MIP-2 (20 ng/mL). The data are reported as the mean ± SEM of cells per field. ..P <

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0.001 compared with RPMI; *P < 0.001 compared with RPMI plus MIP-2 and #P < 0.001

compared with NaHS 300 µM plus MIP-2; n = 5. One hour before the sham-operation (Sham),

non-severe (NS) or severe (S) sepsis induction by CLP surgery, mice were given saline (Sal),

NaHS (100 µmoles/kg, sc), Lawesson’s reagent (100 µmoles/kg, sc), GLB (40 µmoles/kg, sc),

GLB (40 µmoles/kg, sc) plus NaHS (100 µmoles/kg, sc) for another 30 min or GLB (40

µmoles/kg, sc) plus Lawesson’s reagent (100 µmoles/kg, sc) for another 30 min. (B) Six hours

after surgery, neutrophil migration to the infectious focus was determined. The data are reported

as the mean ± SEM x 106 neutrophils/cavity. *P < 0.01 compared to the NS plus Sal group; #P <

0.01 compared to the S plus Sal group; and **P < 0.01 compared to the S plus NaHS group

(ANOVA, followed by Bonferroni’s test, n = 5). (C) Survival rates were monitored over 8 days.

The results are expressed as percent of survival. *P < 0.05 compared to the NS plus Sal group;

#P < 0.01 compared to the S plus Sal group; and **P < 0.01 compared to the S plus H2S group.

Mantel-Cox log-rank test (n = 10-12). Flow cytometry of surface expression of CD11b (D),

CD62L (E) and CXCR2 (F) in blood neutrophils (Gr-1 positive cells) was evaluated 6 h after

surgery. The data are reported as the mean ± SEM of mean fluorescence intensity (MFI) and are

the result of the three independent experiments carried out in triplicate. *P < 0.01 compared to

the NS plus Sal group; #P < 0.01 compared to the S plus Sal group; and **P < 0.01 compared to

the S plus H2S group (ANOVA, followed by Bonferroni’s test, n = 5).

Figure 4. H2S prevented high GRK2 expression in blood neutrophils after severe sepsis via

activation of KATP channels. One hour before sham-operation (Sham), non-severe (NS) or

severe (S) sepsis induction by CLP, mice were given saline (Sal), PAG (50 mg/Kg, ip), GLB (40

µmoles/kg, sc), Lawesson’s reagent (H2S, 100 µmoles/kg, sc) or GLB (40 µmoles/kg, sc) + H2S

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(Lawesson’s reagent 100 µmoles/kg, sc, for another 30 min). Neutrophils were isolated from

total blood 6 hours after surgery, and GRK2 expression was evaluated by immunofluorescence in

Sham (A), NS (B), NS plus PAG (C), NS plus GLB (D), S (E), S plus H2S (F) and S plus GLB

plus H2S (G) groups. These semi-quantitative analyses showed the means of fluorescence

intensity (MFI). The number of animals per groups was 5, and we used 3 slices per animal and

determined the MFI of at least 200 neutrophils per slice. The experiment was repeated three

times. *P < 0.01 compared to the NS plus Sal group; #P < 0.01 compared to the S plus Sal

group; and **P < 0.01 compared to the S plus H2S group (ANOVA, followed by Bonferroni’s

test).

Figure 5. H2S donors enhanced ICAM-1 expression in mesenteric vessels of mice subjected

to severe sepsis by KATP channel activation. One hour before the sham-operation (Sham), non-

severe (NS) or severe (S) sepsis induction by CLP, mice were given saline (Sal), PAG (50

mg/Kg, ip), GLB (40 µmoles/kg, sc), Lawesson’s reagent (H2S, 100 µmoles/kg, sc) or GLB (40

µmoles/kg, sc) + H2S (Lawesson’s reagent 100 µmoles/kg, sc, for another 30 min).

Immunofluorescence staining for ICAM-1 on mesenteric vessels from Sham (A), NS (B), NS

plus PAG (C), NS plus GLB (D), S (E), S plus H2S (F) and S plus GLB plus H2S (G) was

performed. The number of animals per experimental group was 5 (n = 5), and four histological

slices were prepared from each animal. These semi-quantitative analyses showed the means of

fluorescence intensity (MFI) for ICAM-1 on the venular endothelium only. The experiment was

repeated two times. The data represent the mean ± SEM (n = 3). *P < 0.01 compared to the

Sham plus Sal group; **P < 0.01 compared to the NS plus Sal group; and #P < 0.01 compared to

the S plus Sal group (ANOVA, followed by Bonferroni’s test).

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1

HYDROGEN SULFIDE IMPROVES NEUTROPHIL MIGRATION AND

SURVIVAL IN SEPSIS VIA K+

ATP CHANNEL ACTIVATION

Fernando Spiller1, Maria I.L. Orrico

1, Daniele C. Nascimento

1, Paula G. Czaikoski

1,

Fabrício O. Souto3, José C. Alves-Filho

1, Andressa Freitas

1, Daniela Carlos

1, Marcelo

F. Montenegro1, Alberto F. Neto

4, Sergio H. Ferreira

1, Marcos A. Rossi

2, John S.

Hothersall1, Jamil Assreuy

5 and Fernando Q. Cunha

1.

Online Data Supplement

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2

SUPPLEMENT: Table 1

Table E1. Serum cytokine levels 6 h after sepsis induced by cecal ligation and

puncture.

Group

TNF-α (pg/mL) MIP-2 (pg/mL) KC (pg/mL)

Sham-operated 14.15 ±1.96 ND 1.05 ±0.68

Non-severe sepsis 41.65 ±6.46 4.20 ±1.06 583.1 ±165.7

Non-severe sepsis + PAG 103.4 ±9.32 * 221.3 ±81.92 * 1381.0 ±64.68 *

Severe sepsis 87.32 ±7.91 * 636.8 ±68.41 * 1467.0 ±148.8 *

TNF-α, tumor necrosis factor; MIP, macrophage inflammatory protein; KC,

keratinocyte-derived chemokine; ND, not detectable. *P<0.05, compared to non-severe

sepsis group (analysis of variance, followed by Bonferroni’s test).

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SUPPLEMENT: Figure Legends

Figure E1. Plasma H2S levels increase after severe sepsis. (A) Plasma was collected

from mice subjected to non-severe (NS) or severe (S) sepsis after 6 h or from naïve

mice. *P<0.05 compared to naïve mice (ANOVA, followed by Bonferroni’s test, n=5)

(B) NaHS (100 – 1000 µmol/kg) were sc administrated and blood collected 30 min after

this administration. Data are expressed as mean of µM H2S. *P<0.05 compared to doses

of 100 to 300 µmol/kg (ANOVA, followed by Bonferroni’s test, n=5).

Figure E2. Effect of NaHS on MAP of naïve mice and mice with severe sepsis. (A)

Naïve mice were subcutaneous treated with NaHS (100 µmol/Kg) or saline (Sal) and the

mean arterial pressure (MAP) was measured after 6 h. In another set of experiment,

naïve mice were treated intravenously with NaHS (100 µmol/Kg) or Sal and the MAP

monitored for 20 min. *P<0.01 compared to Naive mice treated intravenously with Sal

(ANOVA, followed by Bonferroni’s test, n=5). (B) ∆ of Mean arterial pressure (MAP)

was determined 6 h after severe sepsis induction in mice subcutaneously pretreated (1 h

before surgery) with NaHS (100 µmol/kg, sc) or Sal. #P<0.01 compared to S plus Sal

group (t test, followed by Mann-Whitney U test). Data are reported as ∆ of

means ± SEM of two independent experiments (n=5).

Figure E3. Inhibition of CSE increased the levels of TNF-α and MIP-2 in

peritoneal exudates. Animals were treated with saline (Sal), PAG (50 mg/kg, ip) or

NaHS (100 µmol/kg, sc) 1 hour before sham-operation (Sham), non-severe (NS) or

severe (S) sepsis induction. Six hours after surgery, the peritoneal exudate levels of (A)

TNF-α, (B) MIP-2, and (C) KC in peritoneal exudates were determined by ELISA. Data

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4

are reported as mean ± SEM of two independent experiments (n=5). *P<0.05 compared

to Sham (ANOVA, followed by Bonferroni’s test).

Figure E4. Morphological changes in lung after CLP-induced sepsis. Lungs were

harvested 12 hours after CLP surgery and the tissue sections were stained with

hematoxylin and eosin. Lung sections from mice subjected to sham-operated (A), non-

severe (C) and severe (E) sepsis. Lung sections from mice pretreated (1 h) with PAG

(50 mg/Kg, ip) and subjected to non-severe and severe sepsis are showed in (D) and (F),

respectively. One group of mice were pretreated (1 h) with NaHS (100 µmol/kg, sc) and

subjected to severe sepsis (B). These image of illustrative lung histology are

representative of three independent experiments (n=5). Main panel bars = 100 µm;

insert bars = 25 µm.

Figure E5. Pretreatment of mice subjected to non-severe sepsis with PAG induces

down-modulation of CD62L and CXCR2 and up-regulation of CD11b. One hour

before the sham-operated (Sham), non-severe (NS) or severe (S) sepsis induction by

CLP, mice were given saline (Sal), GLB (40 µmoles/kg, sc) or PAG (50 mg/kg, ip). Six

hours after surgery surface expression of CD11b (A), CD62L (B) and CXCR2 (C) on

blood neutrophils (Gr-1 positive cells) were evaluated by flow cytometry. *P<0.01

compared to NS plus Sal group (ANOVA, followed by Bonferroni’s test, n=5).

Figure E6. H2S donors increased ICAM-1 expression on lung vessels of mice

subjected to severe sepsis. One hour before sham-operation (Sham), and non-severe

(NS) or severe (S) sepsis induction by CLP, mice were given saline (Sal), PAG (50

mg/Kg, ip) or Lawesson’s reagent (H2S, 100 µmoles/kg, sc). Immunofluorescence

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5

staining for ICAM-1 on lung vessels from Sham (A), NS (B), NS plus PAG (C), S (D),

and S plus H2S (E) was performed. The number of animals per experimental group was

5 (n=5) and four histological slices were prepared for each animal. These semi-

quantitative analyses shows the mean fluorescence intensity (MFI) for ICAM-1 on

venular endothelium only. The experiment was repeated two times. *P<0.01 compared

to Sham plus Sal group; **P<0.01 compared to NS plus Sal group; #P<0.01 compared

to S plus Sal group (ANOVA, followed by Bonferroni’s test).

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6

SUPPLEMENT: Detailed Method Sections

Animals

The care and treatment of the animals was based on the Guide for the Care and

Use of Laboratory Animals (1), and the research was approved by the Animal Research

Ethics Committee of the FMRP. Male Swiss mice (22–25 g) were used. The mice were

obtained from the Animal Facility of the FMRP and housed in the Animal Room of the

Department of Pharmacology at 23–25°C with free access to water and food.

Sepsis model

Sepsis was induced by cecal ligation and puncture (CLP model), as described (2).

Mice were anesthetized with tribromoethanol (250 mg/kg, ip), a 1 cm midline incision

was made in the anterior abdomen, and the cecum was exposed and ligated below the

ileocecal junction. The cecum was punctured twice with a 26-gauge needle (for NS

sepsis induction) or twice with an 18-gauge needle (for S sepsis induction), and the

cecum was squeezed to allow its contents to be released through the punctures. Sham

animals underwent identical laparotomy but without cecal puncture. The cecum was

repositioned in the abdomen, and the peritoneal wall was closed. All animals received 1

mL of saline sc immediately after CLP.

Neutrophil migration to the peritoneal cavity

Neutrophil migration was assessed 6 h after induction of sepsis (3). The animals

were killed, and the cells present in the peritoneal cavity were harvested by introducing

1.5 ml of PBS containing 1mM EDTA. Total cell counts from the lavage were

performed with a cell counter (Coulter AC T series analyzer; Coulter Corp., Miami,

USA), and differential cell counts were carried out on cytocentrifuge slides (Cytospin 3;

Shandon Southern Products, Astmoore, UK) stained by the May-Grünwald-Giemsa

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7

(Rosenfeld) method. The results are expressed as the mean ± SEM of the number of

neutrophils per cavity.

Bacterial counts in blood

The bacterial count was determined as previously described (4). Briefly, mice

were killed 6 h after induction of sepsis. To measure bacteremia, blood was collected

under sterile conditions and 10 µl of blood was plated on Muller-Hinton agar dishes

(Difco Laboratories, Detroit, USA) and incubated at 37°C; CFU were analyzed after 12

h. The results were expressed as the mean ± SEM log of CFU per 10 µl of blood.

Intravital microscopy of mesenteric microcirculation

Leukocyte-endothelium interactions (rolling and adhesion) were examined as

previously described (5) in sham-operated or mice subjected to NS or S sepsis by CLP

(mice were pre-treated with saline, NaHS or PAG). Briefly, 2 and 4h after CLP

procedure, mice was anesthetized with tribromoethanol (250 mg/kg) and rolling and

adhesion were analyzed as follow. The mesenteric tissue was exteriorized for

microscopic examination in situ. Animals were maintained on a special board

thermostatically controlled at 37°C, with a transparent platform on which the tissue to

be transilluminated was placed. The preparation was kept moist by irrigating the tissue

with warmed (37°C) Ringer Locke's solution, pH 7.2 to 7.4, containing 1% gelatin. A

500-line television camera was incorporated onto a triocular Zeiss microscope to

facilitate observation of the enlarged image (x 3.400) on the video screen. Images were

recorded on a video recorder with a long distance objective (x 40) with a 0.65 numerical

aperture. The number of leukocyte adhering was determined at 10-min intervals and a

leukocyte was considered to be adherent to the venular endothelium if it remained

stationary for >60 s (6). Adherent cells are expressed as mean ± SEM of the number of

adherent cells per 100 µm2

of venule. Rolling leukocytes were defined as those white

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8

blood cells that moved at a lower velocity than erythrocytes in the

same stream and were

measured at 10 min intervals and are expressed as mean ± SEM of the leukocyte rolling

per min.

Cystathionine Gamma Lyase Assay

CSE activity was measured in total purified leukocytes by a modification of the

method of Steegborn et al. (7) following sham or sepsis induction (2 h). In brief, sample

lysates were prepared by 2 x sonication/freeze/thaw cycles from 106 cells in 100 µl of 5

mM phosphate buffer pH 7.4. Assays (final volume 200 µl) were carried out in 50 mM

phosphate buffer pH 7.4, containing pyridoxal P (0.5 mM) and with or without PAG (1

mM) and samples were pre-incubated at 25°C for 10 min. To start the reaction 5,5’-

dithio-bis(2-nitrobenzoic acid) (DTNB, 5mM) and cystathionine 2 mM were added and

the linear portion of the time course (60 -120 s) was monitored at 412 nm. Activity was

calculated by the OD difference after subtraction of OD change in the PAG containing

sample and calculated using an extinction coefficient Σ412 of 13600 M-1

cm-1

. The

activity of the CSE was expressed as mU/106 cells.

H2S assay by sulfide-sensitive electrode

The blood of mice were collected 6 h after severe sepsis induction or from naïve

mice in tubes with sodium citrate. Measurement of H2S concentration in plasma

samples involved use of a sulfide-sensitive electrode (Innovative Instruments, Inc, FL,

USA). In brief, 1.8 mL of miliQ water was supplemented with 0.2 mL of NaHS

standard solutions (final concentration of the 1, 3, 10, 30 and 100 µM). After each

measurement the electrode was washed with milliQ water. After standard calibration the

samples were measured in the same way. The sample were quantifies from the

calibrated standards.

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9

Determination of Cytokine Levels in the Peritoneal Exudates and Blood

Animals were sacrificed 6 h after sepsis induction, and the peritoneal lavage and

blood collected. Cytokines concentrations were determined by ELISA. The results were

expressed as the mean ± SEM as pg/mL.

Mean arterial pressure

Animals were anaesthetized by ip injection of ketamine (35 mg/Kg) and xilasine

(5 mg/Kg). Left carotid artery was cannulated and connected to a pressure transducer

(TRA 021), and this was coupled to a Power Lab/415 amplifier (MacLab System). One

femoral vein was cannulated for delivering chemicals. A heating pad was used to keep

the body temperature stable at 37°C. After 60 min of equilibration, H2S or saline was

injected in bolus intravenously and MAP was monitored for 20 min.

Isolation of bone marrow neutrophils

Bone marrow cells was collected and neutrophils were isolated by differential

centrifugation on Percoll gradient (8) with minor modifications. Gradient was prepared

in 15-ml polystyrene tube by layering 3 ml each of 72% and 65% Percoll solutions.

After centrifugation at 1200 g for 30 min at 25°C, the cell layer at the 72% upper

interface was collected as the neutrophil fraction. Erythrocytes were removed by lysis in

(NH4Cl 0.075 M) and the remaining neutrophil fractions were washed twice in

HANKS. The pelleted cells were resuspended in 1mL of RPMI-1640 medium (Sigma

Chemical Co., St Louis) and the number of neutrophils determined (90% purity and

95% viable cells).

Chemotaxis Assays

The neutrophil chemotaxis assay was performed using a 48-well modified Boyden

chamber (Neuro Probe Inc., Bethesda, MD, U.S.A.) and 5 µm pore size PVP-free

polycarbonate filters (Nuclepore: Costar Co., Cambridge, MA, U.S.A.). Mice bone

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10

marrow neutrophils (106 cells/mL in RPMI-0.01%) were incubated at 37°C for 1 h

either in the absence (control) or in the presence of GLB or the H2S donor sodium

hydrogen sulfide (NaHS, 30, 100 or 300 µM). In addition, one group was incubated

with GLB (100 µM, 37°C, 30 min) and then NaHS (300 µM) for a further 1 h. After

these respective incubations, neutrophils were allowed to migrate in the Boyden

chamber for 1 h (37°C) in response to RPMI (random migration) or the chemotactic

stimuli MIP-2 (20 ng/mL). Filters were removed, fixed, stained, and neutrophils that

had migrated through the membrane were counted in at least 5 randomly selected fields.

Each sample was assayed in triplicate. Results are expressed as the mean ± SEM

number of neutrophils per field.

Flow Cytometry Analysis of CD11b, CD62L and CXCR2 expression

Flow cytometry was analyzed according to the procedure previously described (9).

Total blood was collected 6 h after CLP and incubated for 30 minutes at 4°C with

fluorescent labeled antibodies: phycoerythrin (PE)-conjugated anti-CXCR2 mAb (1:50;

R&D Systems), phycoerythrin (PE)-conjugated anti-CD62L mAb (1:100; BD

Biosciences), fluorescein (FITC)-conjugated anti-CD11b mAb (1:100; BD

Biosciences) and peridinin chlorophyll protein (PerCP)-conjugated anti-Gr-1 mAb

(1:200; BD Biosciences). The appropriate conjugated mouse isotype control was used in

parallel (BD Biosciences). The cells were then washed twice with lyses buffer (NH4)

and subsequently washed twice in staining buffer (PBS with 5% BSA), fixed in 2%

formaldehyde and analyzed by flow cytometry. The analysis was performed in a

FACSort flow cytometer using CellQuest software (BD Biosciences) by setting a gate

for granulocyte cells derived from a side versus forward scatter dot plot. Neutrophils

were identified by their light-scatter properties and expression of Gr-1 in granulocytes

cells and the expression of CD62L, CD11b and CXCR2 were determined in this

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population. Three to five separate experiments were performed for each analyzed

population of neutrophils and approximately 10,000 gated events were collected in each

analysis.

Immunofluorescence assay for ICAM-1/CD54 or G protein-coupled receptor

kinase 2 (GRK2)

Six hours after CLP, an ICAM-1/CD54 immunofluorescence assay in mesenteric

and lung tissue or GRK2 on neutrophils was carried out according to the procedure

previously described (10). Slices were prepared using 5.0 x104 neutrophils. The

mesenteric and lung tissue was removed, washed in PBS and after freezing serial

sections were mounted on poly-L-lysine covered glass slides. For immunofluorescence

staining, 5 micron frozen tissue sections were fixed with paraformaldehyde (4%) in a

wet chamber at room temperature. The slides were incubated with PBS containing 1%

BSA (PBS-BSA), and then slices were incubated for 1 h with FITC-conjugated anti-

mouse CD54-ICAM-1 (1/200; BD Pharmingen) in PBS-BSA. The neutrophils slices

were then incubated with rabbit anti-mouse GRK2 mAb (1/200; Santa Cruz

Biotechnology) overnight, and then with red-fluorescence Alexa Fluor 594 (goat anti-

rabbit; 1/400; Invitrogen). Subsequently, slides were mounted using 4’,6’-diamidino-2-

phenylindole (Vector Laboratories) and sealed with enamel. The results of qualitative

analysis are expressed as fluorescence intensity of stained venules (x40) or neutrophils

(x40) present in the fluorescence microscopic field.

Statistical analysis

The data are reported as the means ± SEM of values obtained from two to three

independent experiments. We used five mice per experimental group with the exception

of survival analyses in which ten to twelve mice were used. The means value for

different groups were compared by analysis of variance (ANOVA). If significance was

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determined, individual comparisons were subsequently tested with Bonferroni's t test for

unpaired values. Bacterial counts were analyzed by the Mann-Whitney U test. The

survival rate was expressed as the percentage of live animals, and the Fisher´s exact test

was used to determine differences in survival curves. Moreover, the Kolmogorov-

Smirnov test was employed to test for normality. Since the rolling and adhesion results

failed to reach normality, data were log transformed to reach this assumption and then

an ANOVAs were performed with the modified data. A P<0.05 was considered

significant. The data were analyzed using GraphPad Prism version 5.00 for Windows

(GraphPad Software, USA) or SPSS 12.0 for Windows (SPSS, Chicago, IL).

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