Inhibition of Neutrophil Migration by Hemopexin Leads to Increased Mortality Due to Sepsis in Mice

Inhibition of Neutrophil Migration by Hemopexin Leads to Increased

Mortality Due to Sepsis in Mice

Fernando Spiller,1 Carlotta Costa,

5 Fabrício O. Souto,

1 Francesca Vinchi,

5 Fabíola L.A.C.

Mestriner,1 Helen J. Laure,

3,4 José C. Alves-Filho,

1 Andressa Freitas,

1 José C. Rosa,

2,3,4 Sérgio H.

Ferreira,1

Fiorella Altruda,5 Emilio Hirsch,

5 Lewis J. Greene,

2,3,4 Emanuela Tolosano,

5* and

Fernando Q. Cunha1*

From the Departments of 1Pharmacology,

2Cell and Molecular Biology and Pathogenic Agents

and 3Protein Chemistry Center, Faculty of Medicine of Ribeirão Preto, University of São Paulo,

4Hemocenter Foundation of Ribeirão Preto, Ribeirão Preto, SP, Brazil.

5Molecular

Biotechnology Center, University of Torino, Via Nizza 52, 10126 Torino, Italy.

CORRESPONDENCE: Fernando Q. Cunha, Department of Pharmacology, Faculty of Medicine

of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, Monte Alegre 14049–900,

Ribeirão Preto, SP, Brazil. E-mail: fdqcunha@fmrp.usp.br. Tel. 55 16 3602 3324, Fax. 55 16

3633 2301. Emanuela Tolosano, Molecular Biotechnology Center, University of Torino, Via

Nizza 52, 10126 Torino, Italy. E-mail: emanuela.tolosano@unito.it. Tel. 39 011 6706423, Fax.

39 011 6706432.

*Both authors contributed equally to the research.

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: Hx inhibits neutrophil migration

Subject descriptor number: 4.12

Word count: 5172

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

online at www.atsjournals.org.

Scientific Knowledge on the Subject

The reduction of neutrophil migration to the bacterial focus is associated with poor outcome in

sepsis. The mechanism involved in this process is not completely understood, however serum

factors may be responsible for the inhibition of neutrophil migration in severe sepsis.

What This Study Adds to the Field

The abrogation of the acute phase protein hemopexin improves the neutrophil migration to the

focus of infection and survival in sepsis.

of 55 AJRCCM Articles in Press. Published on October 22, 2010 as doi:10.1164/rccm.201002-0223OC

Abstract

Rationale: The reduction of neutrophil migration to the bacterial focus is associated with poor

outcome in sepsis. Objectives: The objective of this study was to identify soluble substances in

the blood of septic mice that inhibit neutrophil migration during the early phases of sepsis.

Results and methods: A pool of serum obtained from mice two hours after the induction of

severe sepsis by cecal ligation and puncture inhibited neutrophil migration. Hemopexin was

identified by mass spectrometry as the serum component responsible for this effect. In sepsis, the

pretreatment of wild type mice with hemopexin inhibited neutrophil migration to the focus of

infection and decreased the survival rate from 87.5% to 50.0%. Hemopexin-null mice submitted

to severe sepsis present normal neutrophil migration and, as a consequence, presented low

bacteremia and an improvement of 40% in survival rate. Moreover, hemopexin inhibited the

neutrophil chemotaxis response evoked by C5a or MIP-2 and induced a reduction of CXCR2 and

L-selectin as well as the up-regulation of CD11b expression in neutrophil membranes. The

inhibitory effect of hemopexin on neutrophil chemotaxis was prevented by serine protease

inhibitors or adenosine-5’-triphosphate (ATP). In addition, serum levels of ATP were decreased

at the early stages of severe sepsis. Conclusion: These data demonstrate for the first time the

inhibitory role of hemopexin on neutrophil migration during sepsis and suggest that the

therapeutic inhibition of hemopexin or its protease activity could improve neutrophil migration

to the focus of infection and survival in sepsis.

Word count: 242

Key-words: neutrophil migration; sepsis; hemopexin; survival; cecal ligation and puncture.

INTRODUCTION

Activation of endothelial cells by chemokines (1) during a localized infection process,

stimulates the rolling and tight adhesion of neutrophils to the endothelium and provides

directional migration cues (2). Once at the site of infection, neutrophils internalize pathogens and

produce toxic substances that kill the invading microorganisms (3, 4). However, when the host

fails to contain the pathogens, they or their products spread to the circulation, resulting in over-

production of inflammatory mediators and impaired immune function (5). We have shown that

the severity of sepsis induced by cecal ligation and puncture (CLP) (6), by Gram-positive (7) or

Gram-negative (8) intraperitoneal (i.p.) bacterial inoculation is associated with inhibition of

neutrophil recruitment to the sites of infection. The inability of neutrophils to migrate results in

an increased number of bacteria in the peritoneal cavity and blood, lung leukocyte sequestration

and high mortality. Furthermore, compared to neutrophils from healthy subjects, neutrophils

obtained from severe septic patients have greatly reduced chemotactic responses (9).

The mechanism involved in the reduction of neutrophil migration observed in severe sepsis

is not completely understood. However, bacteria and/or their products spreading and inducing

activation of the systemic TLRs result in excessive circulating cytokines/chemokines (8, 10)

which could inhibit neutrophil migration. In fact, intravenous (i.v.) administration of LPS (6),

TNF- α or IL-8 inhibits neutrophil migration to the mouse peritoneal cavity. The monoclonal

anti–TNF-α antibody partially prevented the inhibition of neutrophil migration in an

endotoxemia model (11). Moreover, TNFR1/R2 double-knockout mice are more resistant to

severe sepsis (12).

Despite the evidence that cytokines mediate the failure of neutrophil migration, clinical

trials using neutralizing antibodies against cytokines or soluble receptors did not show significant

beneficial effects on septic patients (13). However, other serum factors may also be responsible

for the inhibition of neutrophil migration because massive fresh plasma transfusions restore

neutrophil function in newborns suffering from sepsis (14). In addition, we recently showed that

the acute phase protein alpha-1-acid glycoprotein (AGP) is a serum factor that inhibits neutrophil

migration in severe human sepsis (15).

In experimental mouse sepsis, the serum AGP concentration was significantly increased

only 6 h after the initiation of severe sepsis (unpublished data, Spiller F and Cunha FQ).

However, 2 h after the induction of severe sepsis in mice, there is a decrease in rolling and

adhesion of leukocytes to the endothelium as well as internalization and desensitization of

chemokine receptor CXCR2 on the neutrophil membrane (16), which are essential for neutrophil

migration. Therefore, other serum factors may be involved in the failure of neutrophil migration

during the early phase of severe sepsis and knowledge of these mediators should help in the

management of severe sepsis. Thus, the objective of the present study was to identify soluble

substances other than AGP in the blood of septic mice (15) that inhibit neutrophil migration

during early phases of sepsis.

MATERIALS AND METHODS

Experimental details are provided in the online supplement.

Word count: 502

Neutrophil migration. Neutrophil migration was determined 6 h after CLP or thioglycolate

administration (17).

Sepsis models. Polymicrobial sepsis was induced by CLP model (18). A single puncture was

made using a 21-gauge needle to induce mild sepsis (MS) or four punctures using an 18-gauge

needle to induce severe sepsis (SS). Gram-negative sepsis was induced by Escherichia coli

(ATCC 25922, Bethesda, MD) inoculation.

Neutrophil isolation and chemotaxis assay. Bone marrow neutrophils were isolated by Percoll

gradient (10) and treated (1 h) with 1 µM hemopexin-hemin (Hx-Hem), BSA-Hem or hemopexin

(0.5, 1 and 5 µM). One group was pre-treated (30 min) with ZnDPPG (ZN, 30 µM) before

hemopexin. In another set of experiments, hemopexin (5 µM) was incubated (30 min, 37o) with

phenylmethylsulfonyl fluoride (PMSF, 50 – 200 µM), aprotinin (AP, 2.5 – 10 µM) or adenosine-

5’triphosphate (ATP, 2.5 – 10 µM), then these complexes were incubated with neutrophils for 1

h. After that, neutrophil chemotaxis was performed in response to RPMI (random migration) or

to MIP-2 (20 ng/mL) or C5a (50 or 100 nM).

Flow cytometry analysis. Isolated mouse bone marrow neutrophils were incubated for 1 h with

hemopexin (1, 5 and 10 µM) and stained with antibodies against CD62L, CD11b or CXCR2.

The cells were then analyzed by flow cytometry (FACSort; Becton Dickinson).

Blue-Sepharose chromatography. A pool of 30 ml serum from severe septic mice was

chromatographed on a Blue-Sepharose 6 Fast Flow column to remove most of the albumin (19).

The material with low albumin content was denoted Fraction B-.

HPLC. Fraction B- which contained most of the migration inhibitory activity was submitted to

HPLC on a Vydac C18 column (218TP510, Grace Vydac, Hesperia, CA, USA) using a Shimadzu

Li Chromatography System (Tokyo, Japan).

Native electrophoresis. The HPLC effluent containing neutrophil migration inhibitory activity,

F1 (Figure 2), was submitted to non-SDS polyacrylamide gel electrophoresis (PAGE). Eight µg

of protein of the sample per lane was applied and the protein was localized by Coomassie blue

and eluted by diffusion (20).

Mass spectrometry. Proteins purified by PAGE and a commercial hemopexin sample were heat

denatured and hydrolyzed with Promega modified trypsin (15). Peptide sequences were obtained

by CID-MS/MS. The amino acid sequences of 4 tryptic peptides indicated by * in Table E1

were deduced from the series of b and y fragment ions produced by CID. The masses of tryptic

peptides and fragment ions from CID were used to search the Genebank at NCBI using the

Protein Prospector MS-Fit and MS-Tag software (http://prospector.ucsf.edu/).

Statistical analysis. Data are reported as mean ± SEM (except for survival curves) and are

representative of two or three independent experiments with 5 mice in each group. Differences

between two unpaired groups were compared by Student t test. The mean differences between

three or more groups were compared by ANOVA. When significant differences were identified,

individual comparisons were subsequently tested with Bonferroni’s t-test. Statistical significance

was set at P < 0.05. Survival rates (n=10-12) were expressed as percentages, and a log-rank test

(chi-square test) was used to examine differences between survival curves.

RESULTS

Serum and serum fractions from severe septic mice inhibits neutrophil migration

Mice subjected to severe sepsis (SS) presented 100% mortality (48 h after surgery) and failure of

neutrophil migration to the focus of infection (6 h after surgery), as previously demonstrated (6).

However, mice subjected to mild sepsis (MS) presented ~10% mortality and an efficient

neutrophil migration to the focus of infection (data not shown). The i.v. pretreatment of naïve

mice with serum collected from SS mice 2 h after CLP, significantly reduced thioglycolate-

induced neutrophil migration compared to the mice pretreated with saline or serum from sham-

operated (sham) mice (Figure 1A). The i.v. pretreatment of naïve mice with serum from MS

mice did not significantly inhibit neutrophil migration (data not shown). The next experiments

were designed to identify the soluble substances in the serum from SS mice that inhibit

neutrophil migration.

Serum from SS mice was filtered through an affinity column to remove albumin (Blue-

Sepharose column). Figure 1A shows that most of the neutrophil migration inhibitory activity

was recovered in the fraction that was not adsorbed to the resin (B-). This fraction with low

albumin content (B-) was then filtered through a HPLC C18 column. A typical UV absorbance

profile of fraction B- is shown in Figure 1B. Neutrophil migration inhibitory activity was

detected only in Fraction 1. When a portion of Fraction 1 was submitted to PAGE under native

non-denaturing conditions (Figure 1C), the neutrophil migration inhibitory activity was

demonstrable only in the diffusates of band 3 (B3) and band 4 (B4) (Figure 1A). In contrast, the

part of the gel that did not stain for protein (GEL) did not inhibit neutrophil migration.

Mass spectrometric analysis of the tryptic peptides identified hemopexin as the protein in

fractions B3 and B4. The peptide mass fingerprint of fractions B3 and B4 contained nine

peptides with m/z values that were less than 0.3 mu different from the values expected on the

basis of the amino acid sequence of hemopexin (Figure E1 and Table E1). Four peptides were

submitted to CID-MS/MS and their amino acid sequences were deduced from the ion fragment

pattern (Table E1 and Figure E2). Moreover, the tryptic mass fingerprint obtained from a

commercial sample of hemopexin did not differ significantly from that obtained from fraction B3

or B4 (data not shown). The apparent molecular mass measured by SDS-PAGE of bands B3 and

B4 were ~57 kDa and ~69 kDa (Figure E1C), respectively, as calculated with program Image J

1.37v (National Institutes of Health, USA). The molecular mass of hemopexin is reported to be

~60 kDa (20). A monoclonal antibody against hemopexin detected this protein in fractions B3

and B4 by Western analysis (Figure E1D).

Hemopexin inhibits neutrophil migration and increases mortality in sepsis

Mass spectrometry indicated that hemopexin was the migration inhibitory protein in SS

serum. We used commercial hemopexin to confirm the inhibitory action of hemopexin on

neutrophil migration. Figure 2A shows that i.v. pretreatment of mice with different amounts of

hemopexin significantly inhibited the neutrophil migration stimulated by thioglycolate. BSA

used as a negative control, as expected, did not inhibit neutrophil migration. In addition, serum

collected from hemopexin-null mice 2 h after severe sepsis did not significantly inhibit

neutrophil migration, in contrast to serum collected from wild type mice under the same

conditions from wild type mice (Figure 2A).

To determine the role of hemopexin in sepsis, mice were treated with commercial

hemopexin (10, 30 or 150 µg/mouse, i.v.) 30 min before the induction of mild sepsis. As shown

in Figure 2B, hemopexin treatment resulted in significantly reduced neutrophil migration to the

focus of infection compared to wild type mice treated with saline and subjected to the same

septic stimulus (Figure 2B). The inhibition of neutrophil migration induced by hemopexin was

accompanied by an increased mortality rate of MS mice. Figure 2C shows that the survival rate

of mice submitted to MS and pretreated with saline was 100% 3 d after surgery and 90%

thereafter. The i.v. pretreatment with 10 or 30 µg/mouse hemopexin of MS mice reduced the

survival rate to ~65% 3 d after sepsis. The reduction of the survival rate was more evident when

the MS mice were pretreated with 150 µg/mouse of hemopexin. In this experimental condition

the survival rate was ~50% 3 d after sepsis. Post-treatment (6 h after CLP) with hemopexin (150

µg/mouse) also decreased the survival rate in MS mice (Figure E3).

Moreover, pretreatment of MS hemopexin null-mice with hemopexin (150 µg/mouse, i.v.)

also inhibited the neutrophil migration to the focus of infection (Figure E4A). We also evaluated

the levels of hemopexin after administration of this protein in hemopexin-null mice by ELISA

assay. As showed in Figure E4B the levels of hemopexin was not detected in serum from

hemopexin-null mice pretreated with saline. However, 6 h after i.v. administration of hemopexin

(15 – 300 µg/mouse) we were able to detected circulating levels of this protein in naïve and MS

hemopexin-null mice.

Hemopexin-null mice are more resistant to sepsis

In the previous paragraph we demonstrated the detrimental role of hemopexin in sepsis. To

further investigate this issue, CLP was performed in hemopexin-null mice. The data in Figure

2D show that hemopexin-null mice subjected to SS had a significant increase of neutrophil

number in the peritoneal cavity 6 h after surgery when compared to wild type mice submitted to

the same SS stimulus. The pretreatment (30 min) of hemopexin null-mice subjected to SS with

hemopexin (150 µg/mouse, i.v.) abrogated this improvement of neutrophil migration to the focus

of infection. During MS, both strains presented a similar effective neutrophil recruitment to the

peritoneal cavity 6 h after surgery. However, when neutrophil migration was evaluated 4 h after

MS induction, we observed that hemopexin-null mice had a significantly higher amount (P <

0.05, n=5) of neutrophils in the peritoneal cavity (7.86 ± 0.97 x106 neutrophils/cavity) compared

to wild type mice (3.92 ± 0.82 x106 neutrophils/cavity). The levels of the cytokines IL-1β, TNF-

α, IL-6 and IL-10 in the peritoneal cavity 6 h after the induction of sepsis were similar in both

mouse strains (Figure E5).

We determined the bacterial load in the peritoneal cavity and blood 6 h after CLP. As

shown in Figure 2E, SS hemopexin-null mice exhibited low-levels of bacteria in blood when

compared to SS wild type mice. The infectious focus of hemopexin-null mice also contained

low-levels of bacteria (data not shown). In MS both mouse strains showed similar bacterial

contents.

We also evaluated the systemic inflammatory response of hemopexin-null and wild type

mice to CLP. We first determined the serum levels of TNF-α 6 h after sepsis induction. We

confirmed that the levels of TNF-α were increased in wild type mice during severe sepsis when

compared to non-severe sepsis. However, in hemopexin-null mice with SS, the increase of TNF-

α was not observed (Figure 2F). Furthermore, leukocyte sequestration (measured by MPO

activity) in lung tissue 3 h after sepsis was significantly higher in wild type SS mice than in sham

or MS mice (Figure 2G). This increase in lung neutrophil sequestration was not observed in

hemopexin-null mice with SS.

Finally, we evaluated the mortality of wild type and hemopexin-null mice undergoing MS

and SS induced by CLP. As shown in Figure 2H, both mouse strains subjected to MS presented

survival rates of ~80% throughout the 7 d observation period. In contrast, 100% of wild type

mice subjected to SS died within 2 d, whereas about 40% of hemopexin-null mice subjected to

SS survived throughout the 7 d observation period. Interesting, when hemopexin-null mice were

pretreated with hemopexin (150 µg/mouse, i.v.) all mice died within 2 d after severe sepsis. In

another model of sepsis induced by i.p. inoculation of E. coli ( 2 x108 CFU/mouse), the survival

rate of wild type mice was 48% over a 7 d observation period, as opposed to 86% for

hemopexin-null mice. When wild type mice were pretreated with hemopexin all mice died within

1 d after E. coli inoculation (Figure E6A).

Confirming the anti-inflammatory role of hemopexin, the Figure E6 showed that

hemopexin (150 µg/mouse, i.v.) pre-treatment inhibited LPS-induced leukocyte infiltration in

lungs and the mortality (from ∼100% to ∼30%) in mice. This dose of LPS (5 mg/kg, i.p.) induces

a systemic inflammatory response which is responsible for the low survival of the animals.

Serum hemopexin concentration during sepsis

It has been described that serum levels of hemopexin are up-regulated during an infection

process (21), however, it was not fully addressed in sepsis. As showed in Figure 3A, serum

levels of hemopexin were significantly down-regulated 6 h after severe CLP; however, 12 h after

sepsis induction, there is a significantly increased in the concentration of hemopexin only in

mice subjected to MS. In contrast, the mRNA expression of hemopexin was increased in MS and

SS sepsis 6 h after surgery, and this up-regulation was sustained at 12 h in SS mice (Figure

E7A). Although the serum levels of hemopexin was not increase during SS, the levels of this

protein was significantly higher in the liver of severe septic mice when compared to sham-

operated mice (Figure E7B). The better-characterized function of hemopexin is to scavenge free

heme, following by its transport to the liver (22). Thus, the absence of the correspondent protein

in the serum could be explained by its high turnover due the transport of heme to the liver. To

confirm this hypothesis, we measured the haptoglobin levels (a known marker of hemolysis) and

the levels of total and free heme. The Figure 3B showed that the levels of haptoglobin were

down-regulated, indicating a scenario of hemolysis during severe sepsis. Moreover, the serum

levels of total (Figure 3C) and free heme (Figure 3D) were significantly increased 6 h and 12 h

after surgery only in SS.

The production of hemopexin is known to be regulated by pro-inflammatory cytokines,

such as IL-6 and TNF-α (20), therefore we evaluated the serum levels of hemopexin after CLP in

TNFR1/R2 double-knockout mice (TNFR1/R2-/-

) and in IL-6-deficient mice (IL-6-/-

). The

hemopexin serum levels 2 h and 6 h after MS and SS sepsis induction was similar in both strains

of mice when compared to wild-type mice (data not shown). However, 12 h after MS induction,

the serum levels of hemopexin was significantly decrease in IL-6-/-

mice when compared to

TNFR1/R2-/-

or wild-type mice (Figure E7C).

Hemopexin inhibits the neutrophil chemotactic response in vitro

Bone marrow cells were collected from wild type and hemopexin-null mice and neutrophils were

isolated for testing the in vitro chemotactic response in a Boyden chamber. The chemotactic

responses of neutrophils from both strains were similar. As shown in Figure 4A, C5a (50 nM)

induced a significant chemotactic response in neutrophils from both wild type and hemopexin-

null mice. There was a significant difference between the responses to 50 and 100 nM C5a,

indicating that the response was dose-dependent. The insert in Figure 4A is a Western blot

analysis of serum and of a bone marrow neutrophil extract from wild type and hemopexin-null

naïve mice. A monoclonal antibody against hemopexin detected hemopexin only in serum but

not in neutrophils from wild type mice or in serum from hemopexin-null mice.

The effect of hemopexin on the chemotaxis of neutrophils from bone marrow of wild type

mice was then evaluated. As heme content in serum is increased during severe sepsis, the effect

of the heme-hemopexin complex on neutrophil chemotaxis was also evaluated. Neutrophils were

incubated at 37°C for 1 h in the absence (control, RPMI) or presence of hemopexin (1 µM),

heme-hemopexin (Hx-Hem, 1 µM) or heme-albumin (BSA-Hem, 1 µM) as a negative control

and then the cells (95% viable by Trypan blue exclusion) were allowed to migrate toward 100

nM C5a. As shown in Figure 4B, hemopexin and Hx-Hem pretreatment resulted in a marked

inhibition of C5a-induced chemotaxis. Pretreatment with BSA-Hem had no effect on chemotaxis

at the concentration and time studied. Similarly, hemopexin also inhibited the chemotaxis of

neutrophils from hemopexin-null mice to C5a (Figure 4C). Pretreatment of WT neutrophils with

hemopexin also induced a marked dose-dependent inhibitory effect on chemotactic response to

macrophage-inflammatory protein-2 (MIP-2) (Figure 4D). Moreover, pretreatment (30 min) of

neutrophils with the nonspecific inhibitor of heme oxygenase (HO), zinc deuteroporphyrin 2,4-

bis glycol (ZnDPBG) was not able to block the inhibitory effect of hemopexin on neutrophil

chemotaxis (Figure 4E).

Several studies have provided data demonstrating a protease activity for plasma

hemopexin. Hemopexin purified from human plasma or the recombinant protein was able to

induce transient protein leakage after contact with the rat kidney ex vivo and showed protease

activity that could be inhibited with various serine protease inhibitors or ATP in vitro (23, 24).

To test the role of hemopexin protease activity in the inhibition of neutrophil chemotaxis, we

pre-incubated (30 min, 37°C) hemopexin with the serine protease inhibitors

phenylmethylsulfonyl fluoride (PMSF, 50 – 200 µM) or aprotinin (AP, 2.5 – 10 µM) or with

ATP (2.5 – 10 µM), and then these complexes were added to neutrophil for 1 h. After that,

chemotactic response of neutrophils to MIP-2 was determined. As showed in Figure 4F, PMSF,

AP or ATP significantly prevented the inhibitory effect of hemopexin in neutrophil chemotaxis.

In addition, Figure 4G showed that, when compared to Sham or MS mice, serum from severe

septic mice had a significant decrease in the levels of ATP 2 h after surgery; condition that could

induce the protease activity of hemopexin in vivo.

We isolated neutrophils from TLR4- and MyD88-null mice to determine if the inhibitory

effect of hemopexin on neutrophil chemotaxis was mediated by contamination with LPS in the

sample. Hemopexin (1 or 5 µM) also inhibited the MIP-2-induced chemotaxis of neutrophils

from TLR4- or MyD88-null mice. As expected, LPS inhibited MIP-2-induced chemotaxis of

neutrophils from wild type mice, but was ineffective on neutrophils from TLR4-/- mice (Figure

The leakage of intracellular lactate dehydrogenase (LDH) into the extravascular medium

is an indicator of cell death due to cell membrane damage (25). Therefore we measured the LDH

concentration in the supernatant of neutrophils treated with hemopexin. Neutrophils (1x106/mL)

incubated (4 h, 37oC) with all doses of hemopexin showed similar levels of LDH on supernatants

compared with neutrophils incubated with RPMI. However, neutrophils incubated with RPMI

and subjected to liquid nitrogen lysis showed an ~85% increase of LDH in the supernatant when

compared to cells incubated with RPMI without nitrogen lysis (data not shown). The incubation

of hemopexin (5 µM and 10 µM for 4 h at 37oC) with neutrophils also did not induce the

membrane expression of annexin V or propidium iodide (PI) accumulation in the cells when

compared to control neutrophils (Figure E9).

Hemopexin down-modulates CXCR2 and L-selectin and up-modulates CD11b expression

in neutrophils

In vitro studies have demonstrated that the down-regulation of CXCR2 is responsible for the

reduced neutrophil response to chemokines that bind to CXCR2 (26). Moreover, down-

regulation of CXCR2 on the neutrophil surface is an important event for the failure of neutrophil

migration to the focus of infection during severe sepsis (15). Therefore, we tested whether in

vitro hemopexin neutrophil stimulation leads to CXCR2 down-modulation. Flow cytometric

analysis showed that pretreatment with hemopexin (1, 5 and 10 µM) significantly decreased the

expression of CXCR2 on neutrophils (Figure 5A). Moreover, hemopexin also induced

neutrophil L-selectin shedding (Figure 5B) and up-modulation of CD11b (Figure 5C). All three

effects were dose dependent. L-selectin shedding and increased expression of CD11b on

circulating neutrophils have been used as predictors of severity for septic patients (28, 29).

DISCUSSION

The failure of neutrophil migration to the focus of infection may contribute to the high mortality

rate of severe sepsis (6-8). Several lines of evidence have shown that serum factors induced in

the host during the systemic inflammatory response syndrome (SIRS) are responsible for the

inhibition of neutrophil functions such as migration and oxidative burst (13, 14, 29). In a search

for serum mediators of the inhibition of neutrophil migration during sepsis, we demonstrated

here that the acute phase protein hemopexin participates in this process. First, we showed that

serum collected from wild type mice 2 h after severe sepsis inhibited the thioglycolate-induced

neutrophil migration to the mouse peritoneal cavity. Hemopexin was isolated from this serum

and the purified protein inhibited thioglycolate-induced neutrophil migration to the peritoneal

cavity, an activity also demonstrable for commercial hemopexin. In a mild CLP model of sepsis,

hemopexin inhibited neutrophil migration to the focus of infection and increased the mortality

rate. As expected, hemopexin-null mice subjected to severe sepsis did not present failure of

neutrophil migration to the focus of infection. Consequently, these mice presented less

bacteremia and an improved survival rate when compared to wild type mice. When hemopexin-

null mice were treated with hemopexin before severe CLP, their survival rate as well as

recruitment of neutrophils to the peritoneal cavity, were comparable to what observed in severe

septic wild-type animals. Moreover, hemopexin inhibited neutrophil chemotaxis in vitro and

induced down-modulation of CXCR2 and CD62L, and up-modulation of CD11b expression in

neutrophils. Additionally, we demonstrated that the inhibitory effect of hemopexin under

neutrophil chemotaxis depends on its protease activity, since serine protease inhibitors or ATP

inhibited this process. Thus, the present study has demonstrated for the first time a negative role

for hemopexin in sepsis.

The methods used to isolate hemopexin, i.e., blue Sepharose, C18-HPLC, and PAGE were

standard. However, the difficulty in quantifying the biological response to hemopexin made it

impossible to monitor the recovery of activity of the active substance at each purification step or

to improve the recovery of any step. We recovered ~300 µg of hemopexin from 30 ml of serum,

which is very low, considering that the blood of septic mice contains ~0.50 mg/ml hemopexin.

As is the case for many acute phase proteins, the serum concentration of hemopexin could

increases during infection (21, 30). It is a 60-kDa plasma glycoprotein with a high binding

affinity to heme that forms a 1:1 molar complex. After heme binding, hemopexin undergoes a

conformational change permitting interaction with a specific receptor, expressed mainly on the

hepatocyte membrane, and then is internalized (20, 31). Hemopexin is the main protein

responsible for the transport of intravascular heme, thus preventing both free heme-catalyzed

oxidative damage and heme-bound iron loss (20). Here, for the first time, we demonstrated the

course of serum hemopexin levels during sepsis. Hemopexin mRNA and protein level in the

liver strongly increased after severe sepsis whereas the serum protein level decreased, suggesting

a rapid turnover caused by intravascular hemolysis. This is in agreement with what occurs in

many other pathologic conditions associated to hemolysis including trauma,

hemoglobinopathies, hemorrhages, malaria (22). In addition, our data suggested that IL-6 is the

main regulator of hemopexin production in sepsis, since the IL-6-/-

mice showed lower levels of

hemopexin then wild type mice mice.

We showed here the inhibitory effect of hemopexin on neutrophil migration. When

hemopexin was administered by the i.v. route to mice subjected to mild sepsis, neutrophil

migration to the focus of infection was inhibited. Consequently, control of infection was

impaired, with increased mortality of the mice. In another model of a local sterile inflammation,

hemopexin also inhibited the thioglycolate-induced neutrophil migration to the peritoneal cavity.

Moreover, hemopexin-null mice subjected to severe sepsis presented a significant increase in

neutrophil content at the focus of infection when compared to wild type mice and were more

resistant to sepsis. Of note, in apparent contradiction with these findings, hemopexin null-mice

are more susceptible to heme overload or intravascular hemolysis (30-32). Moreover, in the

present study, hemopexin protected animals from LPS-induced mortality. These models have

similar physiopathology events. The oxidative stress, endothelial cell and leukocyte activation

are the main factors responsible for the tissue injury (32-34). These alterations are triggered

mainly by free heme in heme-overload and intravascular hemolysis models and by LPS in LPS-

induced shock, respectively. In the first two models hemopexin, by sequestering heme, prevents

endothelial and tissue damage. In LPS-induced shock the ability of hemopexin to inhibit

neutrophil migration prevents the neutrophil accumulation in vital organs, such as in lung. In

contrast, the efficient neutrophil migration observed in hemopexin-null mice after the induction

of severe sepsis improved the control of infection, as indicated by the reduction of the CFU

content at the focus of infection and in blood. Bacteria and their products in the blood stream

could induce the activation of TLRs and, therefore, increase the serum levels of cytokines and

trigger leukocyte activation, which accumulated in the lung and other specific organs (8, 35).

Low serum TNF-α levels and the reduction of lung leukocyte sequestration probably account for

the improvement of survival observed in hemopexin-null mice after sepsis.

The higher amounts of neutrophil in the infection focus observed in hemopexin-null mice

undergoing sepsis when compared to wild type mice, was not due to a higher concentration of

cytokines at the site of infection, since both mouse strains presented similar levels of TNF-α, IL-

1β, IL-6 and IL-10 in the exudate. Nevertheless, neutrophil chemotaxis was inhibited when

hemopexin was incubated with neutrophils from wild type or from hemopexin-null mice. Of

note, we observed similar chemotactic response of neutrophils from the bone marrow of wild

type and hemopexin-null mice. Together these results suggest that the mainly effect of the

hemopexin is on the neutrophil locomotion. In addition, Suzuki et. al. (36) demonstrated that

hemopexin inhibits PMA-induced neutrophils adhesion to fibrinogen- and serum-coated surfaces

(37). There is also evidence that hemopexin inhibits the production of cytokines by LPS-

stimulated macrophages (38). However, it seems that it does not happen in sepsis context;

because the level of cytokine production in the focus of infection in hemopexin-null mice was

similar to those in WT mice.

The inhibition of chemotaxis was observed with both hemopexin and the Hx-Heme

complex, suggesting that this effect is independent of the low-density receptor-related protein

(LRP/CD91) present in these cells, since hemopexin alone has low affinity for this receptor.

LRP/CD91 has been reported to internalize Hx-Heme complexes (31). Heme is rapidly

catabolized by HO that opens the porphyrin ring, producing biliverdin and carbon monoxide

(CO) and releasing iron that can then be bound by ferritin (39). It has been shown that the

activation of HO by LPS or hemin with consequent CO production inhibits the neutrophil

chemotactic response and that HO inhibitors prevent this inhibition of the chemotactic response

(Freitas A and Cunha FQ, unpublished data). However, the nonspecific inhibitor of HO did not

block the hemopexin effect on neutrophil chemotaxis, suggesting that the inhibitory effect of

hemopexin is independent of the CD91/HO/CO pathway.

As mentioned above, hemopexin had protease activity which could be inhibited by serine

proteases inhibitors or ATP (24). Here we demonstrated that the co-incubation of hemopexin

with serine protease inhibitors or ATP prevented the reduction of neutrophil chemotaxis induced

by hemopexin. Moreover, the serum level of ATP was decreased in mice with severe sepsis.

These data suggest that severe sepsis triggers an inhibitory activity of hemopexin, likely through

the enhancements of its protease activity and/or decrease the efficiency of their endogenous

inhibitor. This hypothesis may explain why only the septic serum inhibited the neutrophil

migration. Moreover, an intriguing find of the present study was that the inhibitory effect of

human hemopexin upon neutrophil migration is achieved with a very low dose (∼ 8-10 times less

than the physiologic concentration in mice). As human hemopexin behaves like the hemopexin

found in septic mice, it is possible that the human protein is more efficient in inhibiting

migration and/or that the purification procedure from human serum unmasks the inhibitory

effect.

Chemokines coordinate the migration and chemotaxis of leukocytes by binding to specific

chemokine receptors. CXCR2 plays a central role in the recruitment of neutrophils from the

circulation to the site of inflammation (40). Impaired neutrophil migration during sepsis is

correlated with early down-regulation of CXCR2 expression on circulating neutrophils (15). We

have identified hemopexin as a mediator of this phenomenon, since hemopexin induced the

down-regulation of CXCR2 on neutrophils. Interestingly, this decreased CXCR2 expression was

accompanied by reduced chemotaxis to MIP-2. Moreover, hemopexin induced shedding of L-

selectin and up-regulation of CD11b on neutrophils. These findings suggest that the circulating

hemopexin during sepsis could promote the activation of circulating neutrophils and, therefore

inducing the down-regulation of CXCR2 and L-selectin and up-regulation of CD11b and, as a

consequence the inhibition of neutrophil migration to the focus of infection. In accordance, the

protease activity of hemopexin is able to down-regulate angiotensin II receptor-1 in human

monocytes, endothelial cells, and rat aortic rings (41).

The present study provides evidence for another example of an acute phase protein with

anti-inflammatory effects. It has been shown that C-reactive protein (42), serum amyloid A (43),

AGP (14), pentraxin 3 (44) and now hemopexin inhibit neutrophil migration/chemotaxis. The

anti-inflammatory action of acute phase proteins could be beneficial by limiting tissue neutrophil

infiltration in pathological conditions with SIRS without a focus of infection. However, in sepsis,

the overwhelming anti-inflammatory response in the early phase of sepsis inhibits neutrophil

migration and, consequently, impairs bacterial killing. Thus, therapeutic inhibition of acute phase

proteins such as hemopexin, AGP, C-reactive protein and serum amyloid A in the early phase of

sepsis could improve neutrophil migration to the focus of infection and, as a consequence,

improve the survival rate.

Acknowledgments

We thank Giuliana B. Francisco for technical assistance.

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Figure Legends

Figure 1. Serum from severe septic mice and their fractions inhibits the neutrophil

migration. (A) Neutrophil migration to the peritoneal cavity of mice. Six hours after injection of

thioglycolate (3%, 500 µl/cavity, i.p.) the cells in the peritoneal cavity were collected and

neutrophils were counted (see Methods). The test substances were administered i.v. 30 min

before thioglycolate. Data are reported as mean ± SEM x106 neutrophils/cavity (n=5) and are the

result of three independent experiments. Data were analyzed by ANOVA followed by

Bonferroni`s t-test. Serum from sham-operated (Sham) or severe sepsis (SS) mice was injected at

9.6 mg protein/mouse. The protein from the serum of septic mice that adsorbed to the Blue-

Sepharose column (B+) and those that did not adsorb (B-) were injected i.v. at 7.40 and 2.05 mg

protein/mouse, respectively. *P < 0.001 compared to the Sal + Thioglycolate or B+ plus

Thioglycolate groups. The effluent fractions of HPLC (F1 – F4) were injected i.v. at 60 µg

protein/mouse. **P < 0.01 vs Sal plus Thioglycolate group. Diffusates of gel bands 1 to 4 were

injected i.v. at 0.41 µg protein/mouse. The gel control (GEL) was a diffusate from a portion of

the gel which was free of protein. #P < 0.05 compared to Sal or GEL. Sal = Saline. (B) HPLC

profile of serum fraction B-. The fraction of the septic serum pool with low albumin content (B-)

was applied to a C18 column and eluted with a linear gradient of acetonitrile in TFA as described

in Methods. The effluent was combined in four pools as indicated on the horizontal line and the

neutrophil migration inhibiting activity of each pool is shown in panel A. (C) Native gel

electrophoresis diffusates from HPLC fraction 1 (F1).

Figure 2. A commercial sample of plasma hemopexin inhibits neutrophil migration and

hemopexin-null mice are more resistant to severe sepsis. (A) Serum collected 2 h after the

induction of severe sepsis from wild type mice (Wt, 9.50 mg protein/mouse) or from hemopexin-

null mice (Hx-/-, 9.50 mg protein/mouse) and bovine serum albumin (BSA, 30 µg/mouse) or

commercial hemopexin (Hx, 3 – 30 µg/mouse) was injected i.v. 30 min before thioglycolate

(3%, 500 µl/cavity, i.p.). Six hours after thioglycolate administration the neutrophils in the

peritoneal cavity were counted (see Methods). Data are reported as mean ± SEM x106

neutrophils/cavity and are the result of three independent experiments. *P < 0.01 compared to the

Sal plus Thioglycolate group; ** P < 0.05 compared to the Serum Wt plus Thioglycolate group.

The inset in panel A is a Western blot analysis of serum samples from Wt and Hx-/- mice with

severe sepsis; a monoclonal antibody against Hx was used for detection. We used 10-12

mice/group for survival analysis and 5 mice/group for the other data. A log-rank test (chi-square

test) was used to examine differences between survival curves and the other data were analyzed

by ANOVA followed by Bonferroni`s t-test. Commercial Hx (3 -150µg/mouse, i.v.) was injected

30 min before the induction of mild sepsis (MS) by cecal ligation and puncture (CLP). Six h

later, neutrophil migration to the focus of infection was determined (B) and survival rates were

determined up to 7 days after surgery (C). *P < 0.05 compared to the Sal plus MS group. MS

and severe sepsis (SS) were induced in Hx-/- and Wt mice. Commercial Hx (150µg/mouse, i.v.)

was injected 30 min before the induction of SS in Hx-/- mice. Six h after surgery the neutrophil

(D) and bacterial content in blood (E) and serum TNF-α levels (F) were determined. Leukocyte

sequestration in lung (G) was determined 3 h after surgery with a myeloperoxidase assay and the

survival rates (H) were also determined; *P < 0.05 compare to MS Wt group; **P <0.05

compare to SS Wt group.

Figure 3. Serum levels of hemopexin, haptoblogin and total and free heme during sepsis.

Serum were collected from naïve mice or from mice subjected to mild sepsis (MS), severe sepsis

(SS) or sham-operated (Sham) 2, 6 and 12 hours after cecum ligation and puncture (CLP)

surgery. (A) Serum levels of hemopexin (Hx). *P < 0.05 compared to MS or Sham groups; # P <

0.01 compared to all groups. (B) Serum levels of haptoglobin (Hp). *P < 0.05 compared to naive

group; # P < 0.05 compared to MS 12 h group. (C) Serum total and (D) free heme levels; *P <

0.05 compared to Sham or MS. This experiments were repeated 3 times (n=5).

Figure 4. Hemopexin inhibits the neutrophil chemotactic response induced by C5a or MIP-

2. (A) Neutrophils purified from bone marrow of Wild-type (Wt) and from Hemopexin-null (Hx-

/-) mice were submitted to chemotaxis stimulated by C5a (50 or 100 nM) or RPMI (control) in a

Boyden chamber. Data are reported as the mean ± SEM of the number of neutrophils per field

and are the results of three independent experiments carried out in triplicate. *P < 0.001

compared to RPMI; #P < 0.001 compared to 50 nM C5a (analysis of variance followed by

Bonferroni’s test). At the top of panel A is a Western blot analysis of serum and a bone marrow

neutrophil extract from Wt and Hx-/- naïve mice. A monoclonal antibody against hemopexin

(Hx) or vinculin was used for detection. (B) Neutrophils purified from bone marrow of Wt mice

were incubated with RPMI medium, 1 µM Hx, 1 µM Hx-Hem complex or 1 µM bovine serum

albumin-Hem complex (BSA-Hem) for 1 h at 37°C before chemotaxis stimulated by 100 nM

C5a or RPMI (control) in a Boyden chamber. *P < 0.01 compared to RPMI plus RPMI; **P <

0.01 compared to RPMI plus C5a. (C) Neutrophils purified from bone marrow of hemopexin-

null mice were incubated with RPMI medium or 1 µM Hx for 1 h at 37°C before chemotaxis

stimulated by 100 nM C5a or RPMI (control) in a Boyden chamber. *P < 0.01 compared to

RPMI plus RPMI; **P < 0.01 compared to RPMI plus C5a. (D) Hx (0.5 – 5 µM) also inhibited

the neutrophil chemotaxis stimulated by 20 ng/mL MIP-2 in a dose-dependent manner. *P < 0.01

compared to RPMI plus RPMI; **P < 0.01 compared to RPMI plus MIP-2; #P < 0.01 compared

to Hx 0.5 µM plus MIP-2. (E) The inhibitor of heme oxygenase zinc deuteroporphyrin 2,4-bis

glycol (ZnDPBG; ZN 30 µM) did not block the effect of Hx on neutrophil chemotaxis. *P <

0.001 compared to RPMI plus RPMI; **P < 0.01 compared to RPMI plus MIP-2. (F)

Hemopexin (5 µM) were incubated (30 min, 37°C) with phenylmethylsulfonyl fluoride (PMSF,

50 – 200 µM), aprotinin (AP, 2.5 – 10 µM) or with adenosine-5’-triphosphate (ATP, 2.5 – 10

µM). These complexes were added to neutrophils for 1 h and the chemotaxis response to 100 nM

C5a or RPMI (control) in a Boyden chamber was evaluated. *P < 0.01 compared to RPMI plus

MIP-2; **P < 0.01 compared to Hemopexin plus C5a. (G) Levels of ATP were measured in

serum collected 2 h after mild (MS) or severe (SS) sepsis induction or serum from Sham

operated mice.

Figure 5. Hemopexin decreased the expression of CD62L and CXCR2 and increased

CD11b expression on neutrophils. Flow cytometry of CXCR2 (A), CD62L (B) and CD11b (C)

expression on bone marrow neutrophils incubated with RPMI medium or hemopexin (Hx 1, 5

and 10 µM) for 1 h at 37°C. *P < 0.05 compared to RPMI; #P < 0.01 compared to 1 µM Hx.

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.

Figure 1. Serum from severe septic mice and their fractions inhibits the neutrophil migration. (A) Neutrophil migration to the peritoneal cavity of mice. Six hours after injection of thioglycolate (3%, 500 ml/cavity, i.p.) the cells in the peritoneal cavity were collected and neutrophils were counted (see Methods). The test substances were administered i.v. 30 min before thioglycolate. Data are reported as mean ± SEM x106 neutrophils/cavity (n=5) and are the result of three independent experiments. Data were analyzed by ANOVA followed by Bonferroni`s t-test. Serum from sham-operated (Sham) or severe sepsis (SS) mice was injected at 9.6 mg protein/mouse. The protein

from the serum of septic mice that adsorbed to the Blue-Sepharose column (B+) and those that did not adsorb (B-) were injected i.v. at 7.40 and 2.05 mg protein/mouse, respectively. *P < 0.001

compared to the Sal + Thioglycolate or B+ plus Thioglycolate groups. The effluent fractions of HPLC (F1 – F4) were injected i.v. at 60 mg protein/mouse. **P < 0.01 vs Sal plus Thioglycolate group. Diffusates of gel bands 1 to 4 were injected i.v. at 0.41 mg protein/mouse. The gel control (GEL) was a diffusate from a portion of the gel which was free of protein. #P < 0.05 compared to Sal or GEL. Sal = Saline. (B) HPLC profile of serum fraction B-. The fraction of the septic serum pool with

low albumin content (B-) was applied to a C18 column and eluted with a linear gradient of acetonitrile in TFA as described in Methods. The effluent was combined in four pools as indicated on the horizontal line and the neutrophil migration inhibiting activity of each pool is shown in panel A.

(C) Native gel electrophoresis diffusates from HPLC fraction 1 (F1). 166x180mm (300 x 300 DPI)

A commercial sample of plasma hemopexin inhibits neutrophil migration and hemopexin-null mice are more resistant to severe sepsis. (A) Serum collected 2 h after the induction of severe sepsis from wild type mice (Wt, 9.50 mg protein/mouse) or from hemopexin-null mice (Hx-/-, 9.50 mg

protein/mouse) and bovine serum albumin (BSA, 30 µg/mouse) or commercial hemopexin (Hx, 3 – 30 µg/mouse) was injected i.v. 30 min before thioglycolate (3%, 500 ml/cavity, i.p.). Six hours

after thioglycolate administration the neutrophils in the peritoneal cavity were counted (see Methods). Data are reported as mean ± SEM x106 neutrophils/cavity and are the result of three independent experiments. *P < 0.01 compared to the Sal plus Thioglycolate group; ** P < 0.05

compared to the Serum Wt plus Thioglycolate group. The inset in panel A is a Western blot analysis of serum samples from Wt and Hx-/- mice with severe sepsis; a monoclonal antibody against Hx was used for detection. We used 10-12 mice/group for survival analysis and 5 mice/group for the other data. A log-rank test (chi-square test) was used to examine differences between survival

curves and the other data were analyzed by ANOVA followed by Bonferroni`s t-test. Commercial Hx (3 -150µg/mouse, i.v.) was injected 30 min before the induction of mild sepsis (MS) by cecal

ligation and puncture (CLP). Six h later, neutrophil migration to the focus of infection was determined (B) and survival rates were determined up to 7 days after surgery (C). *P < 0.05

compared to the Sal plus MS group. MS and severe sepsis (SS) were induced in Hx-/- and Wt mice. Commercial Hx (150µg/mouse, i.v.) was injected 30 min before the induction of SS in Hx-/- mice. Six h after surgery the neutrophil (D) and bacterial content in blood (E) and serum TNF-α levels (F)

were determined. Leukocyte sequestration in lung (G) was determined 3 h after surgery with a myeloperoxidase assay and the survival rates (H) were also determined; *P < 0.05 compare to MS

Wt group; **P <0.05 compare to SS Wt group. 135x219mm (300 x 300 DPI)

Serum levels of hemopexin, haptoblogin and total and free heme during sepsis. Serum were collected from naïve mice or from mice subjected to mild sepsis (MS), severe sepsis (SS) or sham-operated (Sham) 2, 6 and 12 hours after cecum ligation and puncture (CLP) surgery. (A) Serum

levels of hemopexin (Hx). *P < 0.05 compared to MS or Sham groups; # P < 0.01 compared to all groups. (B) Serum levels of haptoglobin (Hp). *P < 0.05 compared to naive group; # P < 0.05 compared to MS 12 h group. (C) Serum total and (D) free heme levels; *P < 0.05 compared to

Sham or MS. This experiments were repeated 3 times (n=5). 160x118mm (300 x 300 DPI)

Hemopexin inhibits the neutrophil chemotactic response induced by C5a or MIP-2. (A) Neutrophils purified from bone marrow of Wild-type (Wt) and from Hemopexin-null (Hx-/-) mice were submitted to chemotaxis stimulated by C5a (50 or 100 nM) or RPMI (control) in a Boyden chamber. Data are

reported as the mean ± SEM of the number of neutrophils per field and are the results of three independent experiments carried out in triplicate. *P < 0.001 compared to RPMI; #P < 0.001

compared to 50 nM C5a (analysis of variance followed by Bonferroni’s test). At the top of panel A is a Western blot analysis of serum and a bone marrow neutrophil extract from Wt and Hx-/- naïve

mice. A monoclonal antibody against hemopexin (Hx) or vinculin was used for detection. (B) Neutrophils purified from bone marrow of Wt mice were incubated with RPMI medium, 1 µM Hx, 1

µM Hx-Hem complex or 1 µM bovine serum albumin-Hem complex (BSA-Hem) for 1 h at 37°C before chemotaxis stimulated by 100 nM C5a or RPMI (control) in a Boyden chamber. *P < 0.01

compared to RPMI plus RPMI; **P < 0.01 compared to RPMI plus C5a. (C) Neutrophils purified from

bone marrow of hemopexin-null mice were incubated with RPMI medium or 1 µM Hx for 1 h at 37°C before chemotaxis stimulated by 100 nM C5a or RPMI (control) in a Boyden chamber. *P < 0.01 compared to RPMI plus RPMI; **P < 0.01 compared to RPMI plus C5a. (D) Hx (0.5 – 5 µM) also

inhibited the neutrophil chemotaxis stimulated by 20 ng/mL MIP-2 in a dose-dependent manner. *P < 0.01 compared to RPMI plus RPMI; **P < 0.01 compared to RPMI plus MIP-2; #P < 0.01

compared to Hx 0.5 µM plus MIP-2. (E) The inhibitor of heme oxygenase zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG; ZN 30 µM) did not block the effect of Hx on neutrophil chemotaxis. *P < 0.001

compared to RPMI plus RPMI; **P < 0.01 compared to RPMI plus MIP-2. (F) Hemopexin (5 µM) were incubated (30 min, 37°C) with phenylmethylsulfonyl fluoride (PMSF, 50 – 200 µM), aprotinin (AP, 2.5 – 10 µM) or with adenosine-5’-triphosphate (ATP, 2.5 – 10 µM). These complexes were added to neutrophils for 1 h and the chemotaxis response to 100 nM C5a or RPMI (control) in a

Boyden chamber was evaluated. *P < 0.01 compared to RPMI plus MIP-2; **P < 0.01 compared to Hemopexin plus C5a. (G) Levels of ATP were measured in serum collected 2 h after mild (MS) or

severe (SS) sepsis induction or serum from Sham operated mice. 158x219mm (300 x 300 DPI)

Figure 5. Hemopexin decreased the expression of CD62L and CXCR2 and increased CD11b expression on neutrophils. Flow cytometry of CXCR2 (A), CD62L (B) and CD11b (C) expression on

bone marrow neutrophils incubated with RPMI medium or hemopexin (Hx 1, 5 and 10 µM) for 1 h at 37°C. *P < 0.05 compared to RPMI; #P < 0.01 compared to 1 µM Hx. 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. 80x137mm (300 x 300 DPI)

Online Data Supplement

Inhibition of Neutrophil Migration by Hemopexin Leads to Increased

Mortality Due to Sepsis in Mice

Fernando Spiller,1 Carlotta Costa,

5 Fabrício O. Souto,

1 Francesca Vinchi,

5 Fabíola L.A.C.

Mestriner,1 Helen J. Laure,

3,4 José C. Alves-Filho,

1 Andressa Freitas,

1 José C. Rosa,

2,3,4 Sérgio H.

Ferreira,1

Fiorella Altruda,5 Emilio Hirsch,

5 Lewis J. Greene,

2,3,4 Emanuela Tolosano,

5* and

Fernando Q. Cunha1*

SUPPLEMENT: Table 1

Table E1 – Tryptic peptides of hemopexin isolated from the serum of septic mice and

identified by ESI-MS.

*These peptides were sequenced by CID-MS/MS and the protein was identified as

hemopexin. Molecular weight = 51341 and pI = 7.9.

m/z submitted

MH + found

∆ Da

Residues Sequence

975.73 975.55 0.18 103 111 (R) GPDSVFLIK (E)*

1017.75 1017.54 0.22 115 122 (K) VWVYPPEK (K)

1105.76 1105.56 0.20 331 341 (K) GGNNLVSGYPK (R)

1128.78 1128.52 0.26 167 174 (K) WFWDFATR (T) *

1190.81 1190.61 0.20 92 102 (K) NPITSVDAAFR (G) *

1212.86 1212.63 0.22 208 218 (R) FNPVTGEVPPR (Y) *

1505.00 1504.79 0.21 90 102 (R) WKNPITSVDAAFR (G)

1516.86 1516.69 0 .17 270 282 (R) GATYAFTGSHYWR (L)

1814.08 1813.85 0.24 385 400 (K) SGAQATWTEVSWPHEK (V)

SUPPLEMENT: Figure Legends

Figure E1. Peptide mass fingerprint of PAGE bands 3 and 4. Each spectrum contains single

protonated ions obtained by ESI-MS after MaxEnt3 charge deconvolution of proteins from

native electrophoresis bands 3 (A) and 4 (B) (Figure 2B). Four of the ions were identified as

tryptic peptides of Hemopexin (Swiss-prot NP_059067) on the basis of m/z for the ion species

which differed by 0.3 mu or less from the data bank mass assignment (see Table E1). (C) SDS-

PAGE (8-25%) of the proteins diffusates of bands 3 and 4 from native electrophoresis. Standard

0.25 µg of each protein in a low molecular weight calibration kit (Standard), Band 3 and Band 4,

1 µg of each protein diffusates from native electrophoresis Band 3 and Band 4 was applied in

SDS-PAGE. (D) Western blot analysis of commercial hemopexin (Hx), Band 3 and band 4. A

monoclonal antibody against Hx was used for detection.

Figure E2. CID-MS/MS spectrum of ions selected of proteins from native electrophoresis bands

3 and 4. CID-MS/MS of ions m/z 488 [M+2H+] (Mr 974.54) (A); 565 [M+2H+] (Mr 1128.64)

(B); 596 [M+2H+] Mr 1189,60 (C); 607 [M+2H+] Mr 1211,62 (D). The amino acid sequence

deduced from these data are showed in Table E1.

Figure E3. Postreatment with hemopexin increased mortality rate after mild sepsis.

Commercial hemopexin (Hx, 150µg/mouse, i.v.) was injected 6 h after mild sepsis (MS)

induction by cecal ligation and puncture (CLP). Survival rate was evaluated up to 7 days after

surgery (n=10-12).

Figure E4. Hemopexin inhibits the neutrophil migration in mild septic hemopexin-null

mice. Commercial hemopexin (Hx 15 - 150µg/mouse, i.v.) was injected 30 min before the

induction of mild sepsis (MS) by cecal ligation and puncture (CLP) in hemopexin-null mice (Hx-

/-). Six h later, neutrophil migration to the focus of infection (A) and the levels of hemopexin in

serum (B) were determined. The levels of hemopexin were also determined 6 h after

administration of this protein in naïve hemopexin-null mice (panel B). Sal = saline. ND = not

detectable.

Figure E5. Hemopexin-null (Hx-/-) mice and Wild-type (Wt) mice have similar local

inflammatory response after sepsis. IL-1β (A), TNF-α (B), IL-6 (C) and IL-10 (D) levels in

the peritoneal exudate were determined at 6 h after induction of mild (MS) or severe (SS) sepsis

induction by cecal ligation and puncture (CLP). Data are reported as mean ± SEM of pg/mL or

ng/mL (n=5) and represent three independent experiments (data was analyzed by ANOVA

followed by Bonferroni t-test). *P < 0.05 compared to MS Wt group; **P < 0.05 compared to

MS Hx-/- group.

Figure E6. Survival curve after intraperitoneal E. coli inoculation or LPS adminitration.

(A) Wild type (Wt) or hemopexin-null (Hx-/-) mice were treated with saline (Sal) or hemopexin

(Hx, 30µg/mouse, i.v.) 30 min before receiving 2 x108 E. coli CFU/cavity (200µl, i.p.) and

survival was determined up to 7 days after bacterial inoculation. Data are reported as per cent

survival (n=10-12) and are representative of three independent experiments. Hx-/- E. coli + Sal

and Wt E. coli + Hx (30µg/mouse, i.v.) are significantly different from Wt E. coli + Sal (P <

0.05, Mantel-Cox log rank test). Wild type mice were pre-treated (15 min) with hemopexin (Hx,

150 µg/mouse, i.v.) or BSA (bovine serum albumin, 150 µg/mouse, i.v.) and LPS (5 mg/Kg, i.p.)

was administrated. (B) Survival rate was evaluated up to 7 days after LPS administration (n=12).

(C) Leukocyte sequestration in lung was evaluated 3 h after LPS administration (n=5). *P < 0.05

compared to BSA plus Sal (saline); #P < 0.05 compared to BSA plus LPS.

Figure E7. Liver levels of mRNA and protein of hemopexin during sepsis. Liver were

collected from naïve mice or from mice subjected to mild sepsis (MS), severe sepsis (SS) or

sham-operated (Sham) 6 h after cecum ligation and puncture (CLP) surgery. (A) Relative

expression of hepatic mRNA of hemopexin. *P < 0.01 compared to naïve mice. (B) Liver levels

of hemopexin protein. *P < 0.01 compared to naïve or Sham mice. (C) Serum levels of

hemopexin were evaluated 12 h after mild sepsis (MS) induction by cecum ligation and puncture

(CLP) in wild type (Wt), TNFR1/R2 double-knockout mice (TNFR1/R2-/-) or IL-6-deficient

mice (IL-6-/-

). *P < 0.05 compared naïve mice. #P < 0.05 compared MS Wt or TNFR1/R2-/-

mice. These experiments were repeated 3 times (n=5).

Figure E8. Hemopexin inhibits the chemotaxis response of the neutrophils from Toll-like

receptor 4-deficient mice (TLR4-/-, C3H/HeJ) or MyD88-/- mice. (A) Neutrophils purified

from bone marrow of C3H/HePas (TLR4+/+), C3H/HeJ (TLR4-/-), C57/BL6 (Wt) or TLR2-/-

were incubated with media (RPMI), LPS (10 µg/ml) or Hx (1 or 5 µM) for 1 h at 37°C before

chemotaxis stimulated by MIP-2 (20 ng/mL) or RPMI (control) in Boyden chamber. Data are

reported as the mean ± SEM of neutrophils per field and are representative of three independent

experiments carried out in triplicate. *P < 0.01 compared to RPMI plus MIP-2 (analysis of

variance, followed by Bonferroni’s test).

Figure E9. Hemopexin did not induce up-regulation of annexin V or induce propidium

iodide accumulation in neutrophils. Representative dot plot of the annexin V-FITC and

propidium iodide (PI) expression in purified neutrophils from bone marrow of wild type mice.

Neutrophils were incubated 4 h with medium alone (control) or hemopexin (Hx, 5 µM or 10

Methods

Chemicals. Thioglycolate, hemin (Hem), bovine serum albumin (BSA) and May-Grunwald-

Giemsa stain were purchased from Sigma Chemical Company (St. Louis, MO, USA). Human

hemopexin (Hx, >95% by SDS-PAGE) was purchased from Athens Research & Technology,

Inc. (Athens, GA, USA). Lipopolysaccharide (LPS, from Escherichia coli 0127:B8) and

lipoteichoic acid (LTA, from Staphylococcus aureus) were purchased from Difco (Detroit, MI,

USA). All other chemicals were reagent grade or equivalent.

Animals. Research was approved by the Animal Research Ethics Committee of the Faculty of

Medicine of Ribeirão Preto and University of Torino. Breeding pairs of C3H/HeJ mice were

obtained from Jackson Laboratories (Bar Harbor, ME, USA). C3H/HePas mice were obtained

from Institute Pasteur (Paris, France). TLR2-/-

and MyD88-/- mice (129-C57BL/6) were

produced as described previously (1). Hemopexin-null mice were prepared in our laboratory

(University of Turin, Italy). IL-6-/-, TNFR1/R2-/- and C57BL/6 background were obtained from

the School of Medicine of Ribeirao Preto. Male mice (18-22 g) were housed in an animal room

at 23-25oC, with free access to water and food for one day before the experiments.

Neutrophil migration. Thirty minutes after i.v. hemopexin administration, neutrophil migration

was induced by i.p. injection of 500 µl 4% w/v thioglycolate or sepsis was induced by cecal

ligation and puncture (CLP) (2). Neutrophil migration was determined 6 h after CLP or

thioglycolate administration (3). Data are reported as the mean number of neutrophils ± standard

error of the mean (SEM) per cavity (n=5).

Sepsis models. Sepsis was induced by CLP (2). A single puncture was made in the cecum using

a 21-gauge needle to induce mild sepsis (MS) or four punctures using an 18-gauge needle to

induce severe sepsis (SS). Sham-operated animals were submitted to laparotomy without cecal

puncture. Gram-negative sepsis was induced with Escherichia coli (E. coli, ATCC 25922,

Bethesda, MD) inoculation. Lyophilized bacteria were cultured in BHI medium at 37°C (4). The

bacterial suspension (2 x108

CFU/200 µL per mouse) was injected i.p.

Determination of lung tissue myeloperoxidase (MPO) activity. The extent of leukocyte

accumulation in the lung was measured by MPO activity (5). The animals were killed 3 h after

the induction of sepsis or LPS (5 mg/Kg) administration. Lungs were harvested, homogenized

and MPO activity was measured at 450 nm after the addition of tetramethylbenzidine and H2O2.

The results are reported as mean ± SEM of the total number of leukocyte per milligram tissue.

Bacterial count. Mice were killed 6 h after the induction of sepsis. After peritoneal lavage with

sterile PBS/1 mM EDTA and blood collection, aliquots of serial dilutions were plated onto

Müller-Hinton agar dishes (Difco Laboratories) and incubated at 37°C; CFU were counted after

12 h. The results are reported as log CFU per 10 µL.

Neutrophil isolation and chemotaxis assay. An equimolar mixture of hemin with hemopexin or

BSA was prepared (6). Bone marrow

neutrophils were isolated by Percoll

gradient (7).

Neutrophils (106 cells/mL, > 95% homogeneous as determined by May-Grunwald-Giemsa

staining and by high Gr-1 expression by flow cytometry) were treated (1.5 h, 37°C, 5% CO2)

with 1 µM hemopexin-hemin (Hx-Hem), BSA-Hem or hemopexin (0.5, 1 and 5 µM). One group

was pre-treated (30 min) with ZnDPPG (ZN, 30 µM) before hemopexin. In another set of

experiments, hemopexin (5 µM) was incubated (30 min, 37o) with phenylmethylsulfonyl fluoride

(PMSF, 50 – 200 µM), aprotinin (AP, 2.5 – 10 µM) or adenosine-5’triphosphate (ATP, 2.5 – 10

µM), then these complexes were incubated with neutrophils for 1 h at 37°C. Neutrophil

chemotaxis assay (97% viable cells by Trypan blue exclusion) was then performed using a 48-

well modified Boyden chamber (Neuro Probe Inc., Bethesda, MD, USA). Neutrophils were

allowed to migrate in response to RPMI (random migration) or to the chemotactic stimuli MIP-2

(20 ng/mL) or C5a (50 or 100 nM). Data are reported as mean ± SEM number of neutrophils per

field.

Cytokine measurements. Cytokine levels were determined using Luminex

multianalyte

technology. The BioPlex system was used in combination with multiplex cytokine kits (Bio-Rad,

Hercules, CA, USA).

Western blotting. Thirty µg of serum protein or total protein from neutrophil extracts were

separated by 10% SDS-PAGE and analyzed by Western blotting using primary antibodies

against hemopexin (mAb 3D6/E12 produced in our laboratory, University of Torino, Italy) or

vinculin (Sigma-Aldrich). In addition, 1 µg each of Band 3, Band 4, and commercial human

hemopexin was separated on a precast 8-25% PhastGel with PhastGel SDS buffer strips and

transferred to a nitrocellulose filter (Amersham Pharmacia Biotech). The blot was then probed

with a mouse antiserum against hemopexin.

Real-time PCR. RNA was extracted from liver with the MIDI-RNA extraction kit (Qiagen).

Probes for hemopexin and GAPDH were purchased from Applied Biosystems and the

quantitative real-time PCR (qRT-PCR) analysis was performed (8).

Flow cytometry analysis. Mouse bone marrow neutrophils (106

cells) isolated on a Percoll

density gradient (9) were incubated for 1 h (37°C, 5% CO2) with hemopexin (1, 5 and 10 µM),

and stained with R-PE-conjugated rat anti-mouse CD62L (L-selectin) monoclonal antibody (BD

Bioscience), FITC-conjugated rat anti-mouse CD11b monoclonal antibody (BD Bioscience), R-

PE anti–CXCR2 antibody (Santa Cruz Biotechnology) or appropriate isotype controls. In another

set of experiments, the bone marrow neutrophils were incubated with hemopexin (5 and 10 µM)

for 4 h (37°C, 5% CO2) and the cells were stained for annexin V and propidium iodide (PI) by an

apoptosis detection kit (Bender MedSystems). The cells were then analyzed by flow cytometry

(FACSort; Becton Dickinson).

Protein determination. Amino acid composition and total protein were determined by amino

acid analysis using the PTC method (10). The protein content of serum, HPLC and

electrophoresis fractions as well as hemopexin was determined after hydrolysis in 6 N HCl at

1200C for 22 h.

Blue-Sepharose chromatography. A pool of 30 ml serum from severe septic mice was

chromatographed on a Blue-Sepharose 6 Fast Flow column to remove most of the albumin (11).

The material that was not absorbed was combined and denoted Fraction B-. The column was

then washed to strip albumin and other proteins from the resin. This effluent was denoted

Fraction B+.

HPLC. Fraction B- which contained most of the migration inhibitory activity was submitted to

HPLC on a Vydac C18 column (218TP510, Grace Vydac, Hesperia, CA, USA) using a Shimadzu

Li Chromatography System (Tokyo, Japan). The solvents were A (0.1% trifluoroacetic acid,

TFA) and B (80% acetonitrile containing 0.09% TFA). The column was equilibrated with 80 mL

35 % B. A 200 µl sample was applied to the column and a gradient of 1% B/minute was applied

immediately after sample injection. The flow rate was 4.0 ml/min. Effluent absorbance was

monitored at 220 and 280 nm and effluent was combined on the basis of absorbance.

Native electrophoresis. The HPLC effluent containing neutrophil migration inhibitory activity,

F1 (Figure 2), was submitted to non-SDS polyacrylamide gel electrophoresis (PAGE). Eight µg

of protein of the sample per lane was applied and the protein was localized by Coomassie blue

and eluted by diffusion (12).

Mass spectrometry. Proteins purified by PAGE and a commercial hemopexin sample were heat

denatured and hydrolyzed with Promega modified trypsin (13). The mass fingerprint of the

tryptic peptides was obtained with an electrospray triple-quadrupole mass spectrometer, Quattro

II (Micromass, Manchester, UK). Peptide sequences were obtained by collision-induced

dissociation tandem mass spectrometry (CID-MS/MS). All reported spectra are the average of

13-18 scans (2 to 5 sec/scan) and were processed using the Mass Lynx software v.3.3

(Micromass). The amino acid sequences of 4 tryptic peptides indicated by * in Table E1 were

deduced from the series of b and y fragment ions produced by CID. The masses of tryptic

peptides and fragment ions from CID were used to search the Genebank at NCBI using the

Protein Prospector MS-Fit and MS-Tag software (http://prospector.ucsf.edu/).

Analysis of serum ATP levels

The denaturation of sample proteins was performed using 0.6 mol/L perchloric acid. All samples

were then centrifuged (14000 ×g for 10 min) and the supernatants were neutralized with 4.0 N

KOH and clarified with a second centrifugation (14000 ×g for 15 min). Aliquots of 20 µL were

applied to a reverse-phase HPLC system using a 15 cm C18 Shimadzu column (Shimadzu, Japan)

at 260 nm with a mobile phase containing 60 mM KH2PO4, 5.0 mM tetrabutylammonium

chloride, pH 6.0 (buffer A), in 20% methanol (buffer B, 0 to 100% of buffer B in 25 min)

according to a method previously described (14). The peak of ATP were identified by their

retention times and quantified by comparison with standards. The results are expressed by nmol

of ATP per mL of serum.

Quantification of heme levels

Whole blood was collected by cardiac puncture into heparinized tubes and centrifuged (15

minutes, 4°C, 500 g) to obtain the supernatant. The plasma was centrifuged again (10 min, 4°C,

500 g) to remove contaminating red blood cells and passed it through a Centricon YM-3 column

(Millipore) (2 h at 4°C, 3760 g) to remove proteins with MW>3 kDa. Heme and protein-free

heme were quantified using a chromogenic assay according to the manufacturer’s instructions

(QuantiChrom heme assay kit, Bioassay Systems). Heme levels were determined 2, 6 and 12 h

after sepsis induction. The absorbance was read at 400 nm. The results are expressed in µM of

Quantification of hemopexin levels

Hemopexin quantification was performed by ELISA using commercially available kits according

to the respective manufactures specifications (GenWay Biotech).

Statistical analysis. Data are reported as mean ± SEM (except for survival curves) and are

representative of two or three independent experiments with 5 mice in each group. Differences

between two unpaired groups were compared by the Student t test. The mean differences

between three or more groups within an experiment were compared by ANOVA. When

significant differences were identified, individual comparisons were subsequently tested with

Bonferroni’s t-test. Statistical significance was set at P < 0.05. Survival rates (n=10-12) were

expressed as percentages, and a log-rank test (chi-square test) was used to examine differences

between survival curves.

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Peptide mass fingerprint of PAGE bands 3 and 4. Each spectrum contains single protonated ions obtained by ESI-MS after MaxEnt3 charge deconvolution of proteins from native electrophoresis

bands 3 (A) and 4 (B) (Figure 2B). Four of the ions were identified as tryptic peptides of Hemopexin (Swiss-prot NP_059067) on the basis of m/z for the ion species which differed by 0.3 mu or less

from the data bank mass assignment (see Table E1). (C) SDS-PAGE (8-25%) of the proteins diffusates of bands 3 and 4 from native electrophoresis. Standard 0.25 µg of each protein in a low

molecular weight calibration kit (Standard), Band 3 and Band 4, 1 µg of each protein diffusates from native electrophoresis Band 3 and Band 4 was applied in SDS-PAGE. (D) Western blot analysis of commercial hemopexin (Hx), Band 3 and band 4. A monoclonal antibody against Hx was used for

detection. 186x130mm (600 x 600 DPI)

CID-MS/MS spectrum of ions selected of proteins from native electrophoresis bands 3 and 4. CID-MS/MS of ions m/z 488 [M+2H+] (Mr 974.54) (A); 565 [M+2H+] (Mr 1128.64) (B); 596 [M+2H+] Mr 1189,60 (C); 607 [M+2H+] Mr 1211,62 (D). The amino acid sequence deduced from these data

are showed in Table E1. 181x160mm (600 x 600 DPI)

Postreatment with hemopexin increased mortality rate after mild sepsis. Commercial hemopexin (Hx, 150µg/mouse, i.v.) was injected 6 h after mild sepsis (MS) induction by cecal ligation and

puncture (CLP). Survival rate was evaluated up to 7 days after surgery (n=10-12). 78x65mm (300 x 300 DPI)

Hemopexin inhibits the neutrophil migration in mild septic hemopexin-null mice. Commercial hemopexin (Hx 15 - 150µg/mouse, i.v.) was injected 30 min before the induction of mild sepsis (MS) by cecal ligation and puncture (CLP) in hemopexin-null mice (Hx-/-). Six h later, neutrophil

migration to the focus of infection (A) and the levels of hemopexin in serum (B) were determined. The levels of hemopexin were also determined 6 h after administration of this protein in naïve

hemopexin-null mice (panel B). Sal = saline. ND = not detectable. 79x48mm (300 x 300 DPI)

Hemopexin-null (Hx-/-) mice and Wild-type (Wt) mice have similar local inflammatory response after sepsis. IL-1β (A), TNF-α (B), IL-6 (C) and IL-10 (D) levels in the peritoneal exudate were

determined at 6 h after induction of mild (MS) or severe (SS) sepsis induction by cecal ligation and puncture (CLP). Data are reported as mean ± SEM of pg/mL or ng/mL (n=5) and represent three independent experiments (data was analyzed by ANOVA followed by Bonferroni t-test). *P < 0.05

compared to MS Wt group; **P < 0.05 compared to MS Hx-/- group. 179x144mm (300 x 300 DPI)

Survival curve after intraperitoneal E. coli inoculation or LPS adminitration. (A) Wild type (Wt) or hemopexin-null (Hx-/-) mice were treated with saline (Sal) or hemopexin (Hx, 30µg/mouse, i.v.) 30

min before receiving 2 x108 E. coli CFU/cavity (200µl, i.p.) and survival was determined up to 7

days after bacterial inoculation. Data are reported as per cent survival (n=10-12) and are representative of three independent experiments. Hx-/- E. coli + Sal and Wt E. coli + Hx

(30mg/mouse, i.v.) are significantly different from Wt E. coli + Sal (P < 0.05, Mantel-Cox log rank test). Wild type mice were pre-treated (15 min) with hemopexin (Hx, 150 µg/mouse, i.v.) or BSA

(bovine serum albumin, 150 µg/mouse, i.v.) and LPS (5 mg/Kg, i.p.) was administrated. (B) Survival rate was evaluated up to 7 days after LPS administration (n=12). (C) Leukocyte

sequestration in lung was evaluated 3 h after LPS administration (n=5). *P < 0.05 compared to BSA plus Sal (saline); #P < 0.05 compared to BSA plus LPS.

129x146mm (300 x 300 DPI)

Liver levels of mRNA and protein of hemopexin during sepsis. Liver were collected from naïve mice or from mice subjected to mild sepsis (MS), severe sepsis (SS) or sham-operated (Sham) 6 h after cecum ligation and puncture (CLP) surgery. (A) Relative expression of hepatic mRNA of hemopexin. *P < 0.01 compared to naïve mice. (B) Liver levels of hemopexin protein. *P < 0.01 compared to naïve or Sham mice. (C) Serum levels of hemopexin were evaluated 12 h after mild sepsis (MS)

induction by cecum ligation and puncture (CLP) in wild type (Wt), TNFR1/R2 double-knockout mice (TNFR1/R2-/-) or IL-6-deficient mice (IL-6-/-). *P < 0.05 compared naïve mice. #P < 0.05

compared MS Wt or TNFR1/R2-/- mice. These experiments were repeated 3 times (n=5). 129x140mm (300 x 300 DPI)

Hemopexin inhibits the chemotaxis response of the neutrophils from Toll-like receptor 4-deficient mice (TLR4-/-, C3H/HeJ) or MyD88-/- mice. (A) Neutrophils purified from bone marrow of

C3H/HePas (TLR4+/+), C3H/HeJ (TLR4-/-), C57/BL6 (Wt) or TLR2-/- were incubated with media

(RPMI), LPS (10 µg/ml) or Hx (1 or 5 µM) for 1 h at 37°C before chemotaxis stimulated by MIP-2 (20 ng/mL) or RPMI (control) in Boyden chamber. Data are reported as the mean ± SEM of neutrophils per field and are representative of three independent experiments carried out in

triplicate. *P < 0.01 compared to RPMI plus MIP-2 (analysis of variance, followed by Bonferroni’s test).

129x71mm (300 x 300 DPI)

Hemopexin did not induce up-regulation of annexin V or induce propidium iodide accumulation in neutrophils. Representative dot plot of the annexin V-FITC and propidium iodide (PI) expression in

purified neutrophils from bone marrow of wild type mice. Neutrophils were incubated 4 h with medium alone (control) or hemopexin (Hx, 5 µM or 10 µM).

129x56mm (300 x 300 DPI)

Inhibition of Neutrophil Migration by Hemopexin Leads to Increased Mortality Due to Sepsis in Mice

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