TUMOR NECROSIS FACTOR IS NOT ASSOCIATED WITH INTESTINAL ISCHEMIA/REPERFUSION-INDUCED LUNG...

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TUMOR NECROSIS FACTOR IS NOT ASSOCIATED WITHAQ1 INTESTINALISCHEMIA/REPERFUSIONYINDUCED LUNG INFLAMMATION

Alexandre Learth Soares,* Fernando Rodrigues Coelho,* Rodrigo Guabiraba,†

Mamdouh Kamal,† Bernardo Boris Vargaftig,* Lily Li,‡ Jian Li,§

Wothan Tavares de Lima,* and Bernhard Ryffel†

*Institute of Biomedical Sciences, Department of Pharmacology, University of Sao Paulo, Sao Paulo,Brazil; †Laboratory of Molecular Immunology and Embryology, University of Orleans and CNRS UMR

6218, Orleans, France; and ‡Therakos, Department of Immune Cell Therapy, Exton; and §Centocor R&D,Department of Immunobiology, Horsham, Pennsylvania

Received 14 Oct 2009; first review completed 20 Oct 2009; accepted in final form 12 Nov 2009

ABSTRACT—Intestinal ischemia-reperfusion (I/R) injury may cause acute systemic and lung inflammation. Here, werevisited the role of TNF-! in an intestinal I/R model in mice, showing that this cytokine is not required for the local andremote inflammatory response upon intestinal I/R injury using neutralizing TNF-! antibodies and TNF ligandYdeficientmice. We demonstrate increased neutrophil recruitment in the lung as assessed by myeloperoxidase activity and aug-mented IL-6, granulocyte colony-stimulating factor, and KCAQ2 levels, whereas TNF-! levels in serum were not increased andonly minimally elevated in intestine and lung upon intestinal I/R injury. Importantly, TNF-! antibody neutralization neitherdiminished neutrophil recruitment nor any of the cytokines and chemokines evaluated. In addition, the inflammatoryresponse was not abrogated in TNF and TNF receptors 1 and 2Ydeficient mice. However, in view of the damage on theintestinal barrier upon intestinal I/R with systemic bacterial translocation, we asked whether Toll-like receptor (TLR)activation is driving the inflammatory response. In fact, the inflammatory lung response is dramatically reduced in TLR2/4Ydeficient mice, confirming an important role of TLR receptor signaling causing the inflammatory lung response. Inconclusion, endogenous TNF-! is not or minimally elevated and plays no role as a mediator for the inflammatory responseupon ischemic tissue injury. By contrast, TLR2/4 signaling induces an orchestrated cytokine/chemokine response leadingto local and remote pulmonary inflammation, and therefore disruption of TLR signaling may represent an alternativetherapeutic target.

KEYWORDS—Ischemic tissue injury, pulmonary inflammation, cytokines, TNF, TLR

INTRODUCTION

Intestinal ischemia-reperfusion (I/R) injury is a serious and

common clinical entity and can result from different etiologic

factors, including superior mesenteric artery (SMA) occlusion

or hemorrhagic shock (1). This condition may result in severe

local and extensive tissue injury and subsequent distant organ

dysfunction, affecting mainly the lungs. Several studies (2, 3)

point to the important role that intestinal I/R may play in the

triggering of acute respiratory distress syndrome (ARDS), a

syndrome characterized by hypoxemia, reduced lung com-

pliance, fluffy diffuse infiltrates on the chest radiograph, and

the presence of normal pulmonary capillary pressures (4).

Although most patients survive the initial insult that precipi-

tates ARDS, no therapeutic protocol has proven to modify the

course of this condition and mortality remains greater than

40% (5).

Among the mediators that are altered during intestinal I/R,

TNF-! is the most widely studied (6). TNF-! at high levels

leads to the development of an inflammatory response that is

the hallmark of many diseases because the mediator is impli-

cated in acute lung injury and ARDS, and also more chronic

lung diseases such as asthma, chronic bronchitis, and chronic

obstructive pulmonary disease (7, 8).

TNF-! binds to two receptors, the 55-kd and 75-kd recep-

tors, with similar affinity (6). The 55-kd receptor (TNFR1)

mediates cytotoxic and proinflammatory effect, whereas the

75-kd receptor (TNFR2) mediates T-cell proliferation (8). Al-

though there is a well-established link between TNF-! levels

and sepsis, there is still a debate on the role of TNF-! in non-

septic acute inflammatory conditions. In fact, depending on

the model used for the induction of the inflammatory insult,

no significant increase of circulating TNF-! is detected. Kim

et al. (9) showed that the levels of circulating TNF-! did not

change compared with sham controls in a model of hemorrhage

shock, whereas Grotz et al. (10) showed early TNF-! increases

upon intestinal I/R in rats that are primarily caused by the

laparotomy. Chen et al. (11) showed that the Toll-like receptor

(TLR) ligand LPS decreased mesenteric I/R injury through

TNF-! signaling.

The TLR family members recognize pathogen-associated

molecular patterns derived from microbes as well as endoge-

nous ligands from damaged/stressed cells (12). Toll-like recep-

tor 2 and TLR4 are activated not only by LPS and lipoteichoic

1

SHOCK, Vol. 00, No. 00, pp. 00Y00, 2010

Copyeditor: Maria Charissa Paras

Address reprint requests to Bernhard Ryffel, Molecular Immunology and

Embryology, University and CNRS UMR 6218. 45071, Orleans, France. E-mail:

[email protected].

This study was supported by grants from the Fondation de la Recherche Medical

(F.R.M, France); PhenoTec AG, Zug, Switzerland; Immunis SA, Orleans, France;

and Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Sao Paulo,

Brazil.

Supplemental digital content is available for this article. Direct URL citation

appears in the printed text and is provided in the HTML and PDF versions of this

article on the Journal’s Website (www.shockjournal.com).

DOI: 10.1097/SHK.0b013e3181cdc585

Copyright � 2010 by the Shock Society


acid but also by extracellular matrix components (13) and pos-

sibly by heat shock proteins (14). Toll-like receptor 4 has also

been implicated in the priming effect induced by hemorrhagic

shock for lung inflammation (15).

Here, we tested the hypothesis that TNF-! is not required

for the local and remote inflammatory response upon intes-

tinal I/R injury using neutralizing TNF-! antibodies and TNF

ligandYdeficient mice (KO). We report that after intestinal

I/R in mice, several cytokines are upregulated, in particular of

IL-6, granulocyte colony-stimulating factor (G-CSF), and KC,

but TNF-! levels are very little affected. Importantly, neu-

tralization of TNF-! by antibodies did not affect neutrophil

recruitment to the lung, the cytokine response, and intestinal

and pulmonary injury. Furthermore, the inflammatory re-

sponse in the absence of TNF-! or of TNF receptors was not

significantly different from wild-type controls upon intestinal

I/R injury. Finally, we demonstrate that TLR2/TLR4 receptor

signaling plays a critical role in the inflammatory process

after intestinal I/R in mice.

MATERIALS AND METHODS

AnimalsMale C57Bl/6 mice aged 8 to 10 weeks were purchased from Ace

(Philadelphia, Pa) or obtained from the animal care facility at the TransgenoseInstitute (CNRS, Orleans, France). TNF KO and TNFR1/R2 KO (obtainedfrom Jackson Laboratories, Bar Harbor, Me), and TLR2 and TLR4 KO(obtained from Dr Akira, crossed in-house to obtain TLR2/4 KO mice) (16)

FIG. 1. Effects of intestinal I/R on MPO activity in lung (A) and intestine (B) LDH activity in serum (C) and gut (D). Histological example onhematoxylin-eosin slides. Mice were subjected to 45 min of mesenteric artery occlusion followed by 4 h of intestinal reperfusion. Lung samples from ischemicanimal (F and H). Control group consisted of mice that were sham operated (E and G) (n = 7) (hematoxylin-eosin, original magnification �400). E and F,Representative section of a small branch of pulmonary arteriole. G and H, The alveolar structure, where arrows point to polymorphonuclear cells infiltrated in thetissue. Basal values were obtained from naive mice. Data represent mean T SEM from 2 experiments from 5 to 7 animals. *P G 0.05.

2 SHOCK VOL. 00, NO. 00 SOARES ET AL.


used in this study were bred under specific pathogenYfree conditions at theTransgenose Institute (CNRS). Animals were housed, kept in sterile isolatedventilated cages, and used after an approved, reviewed, and monitoredprotocol from the Committee on Care and Use of Laboratory Animals ofCentocor R&D and from the French Government’s ethical and animalexperiment regulations. All animals had access to water and food ad libitumbefore the experiments.

Animal model of acute lung injuryIntestinal I/R was performed as described elsewhere (17). In brief, lapa-

rotomy was performed under anesthesia with ketamine (100 mg/kg, intra-peritoneally) and xylazine (20 mg/kg, intraperitoneally). In the ischemic groupof mice, a microsurgical clip (Vascu-statt no. 1001-531; Scalan Internat,Minn) was applied to occlude the SMA. After 45 min occlusion, the clip wasremoved and intestinal perfusion was re-established. Animals were kept inanesthesia and killed 4 h after intestinal reperfusion by cervical dislocation.For the 24 h time point, the animals were awake after reperfusion andreceived acetaminophen-supplemented drinking water (3.2 mg/mL) to relievepain. Blood samples were collected from the abdominal aorta of anesthetizedmice. The aliquots were centrifuged (170 g, 10 min), and the sera obtainedwere stored at j80-C until further analyses. Sham-operated mice undergoingidentical surgical intervention except for the occlusion of the SMA were usedas control groups. An additional group of nonmanipulated mice (naive) wasadded to obtain basal values of the parameters studied.

Measurement of lactate dehydrogenaseIntestinal tissue damage caused by intestinal I/R releases lactate dehydro-

genase (LDH) into the systemic circulation and was measured according

to Cavriani et al. (17). Briefly, LDH activity was determined in the serumsamples using a commercially available kit (Labtest Diagnostica, BeloHorizonte, Brazil) and expressed as units per microliter. Absorbance wasread at 340 nm on a microplate reader spectrophotometrically (Bio-TekInstruments).

Lung and intestine myeloperoxidase activityMyeloperoxidase (MPO) activity was measured as an index of the presence

of neutrophils. The lungs were perfused via the pulmonary artery with 3 mLof ice-cold pH 7.2 phosphate-buffered saline (PBS). Intestines were rinsedwith ice-cold PBS, and the small intestine was excised and used in theanalysis. This procedure was also used to obtain the samples for cytokineand histological analyses. Samples were prepared according to Goldblumet al. (18). In brief, to normalize the pulmonary and intestinal MPO activityamong the groups, the whole tissue was homogenized with 3 mL/g of PBScontaining 0.5% of hexadecyl-trimethylammonium bromide and 5 mMEDTA, pH 6.0. The homogenized samples were centrifuged at 37,000g for15 min. Samples of lung and intestine homogenates (10 2L) were thenincubated for 10 min with H2O2 and ortho-dianisidine, and the reaction wasstopped by the addition of 1% NaNO3. The absorbance was determined at450 nm using a microplate reader (Bio-Tek Instruments).

HistologyIntestine and lung were fixed in 10% buffered formalin. Tissues were

dehydrated in ethanol and embedded in paraffin. Sections of 5 2m werestained with hematoxylin and eosin for evaluation of pathological changes.The microscopic slides were reviewed and assessed using a semiquantitative

FIG. 2. Cytokines levels in intestine homogenates from mice upon intestinal I/R. Groups of mice were submitted to 45 min of intestinal ischemiafollowed by 4 h of reperfusion. Sham-operated mice were used as controls. Basal values were obtained from naive mice. Cytokine levels were determined inintestine homogenates using multiplex analysis IL-1! (A), IL-6 (B), KC (C), TNF-! (D), G-CSF (E), and MCP-1 (F). Data represent mean T SEM from 2experiments from 5 to 7 animals. *P G 0.05.

SHOCK MONTH 2010 TNF IN INTESTINAL ISCHEMIC INJURY 3


score by two investigators (F.R.C., B.R.) who were blinded to the groups andtime points.

Cytokine measurementCytokines levels were evaluated in serum and in lung and gut homogenates

using a 22-cytokine Linco multiplex kit (LINCO Research, Inc, Mo) with aLuminex instrument (Luminex Corporation, Tex).

In vivo neutralization of TNF-!Rat monoclonal antibody (mAb) with neutralizing activity for murine

TNF-! was generated in house (Centocor R&D, Horsham, Pa) using standardhybridoma technology. The neutralizing property of this antibody has beentested in vitro and in vivo in response to TNF (data not shown). Prior (30 min)to intestinal I/R induction, mice were i.v. injected with 10 mL/kg of a solutioncontaining 2.5 mg/mL of TNF-! antibody (final dose, 25 mg/kg of antibody).A group of mice i.v. injected with 10 mL/kg of PBS 30 min before theintestinal I/R induction was used as the control.

Expression of TLR2 and TLR4 mRNA in lung tissueSamples of lungs were excised and frozen in liquid nitrogen after the

experimental procedure. To harvest RNA, samples were lysed using NucleicAcid Purification Lysis solution (Applied Biosystems, Foster City, Calif).RNA was prepared using ABI PRISM 6100 Nucleic Acid PrepStation (DNasestep included). RNA quality was verified with the 2100 BioAnalyzer (AgilentTechnologies, Palo Alto, Calif). Samples that demonstrated high quality (i.e.,the ratio of 28S rRNA to 18S rRNA, 91.7) and had a minimum of 1 2g ofRNA were submitted to real-time polymerase chain reaction (PCR) analysis.

The expression of TLR2 and TLR4 mRNA was evaluated by real-time PCRusing a TaqMan assay kit, following the manufacturer’s recommendations.

Statistical analysisData are given as mean T SEM. Comparisons between the groups were

made by one-way ANOVA followed by unpaired Newman-Keuls post-test. A4.0 version (2005) of the GraphPad Prism software was used for this purpose.Values of P G 0.05 were considered significant.

RESULTS

Model characterization

To study the inflammatory response upon intestinal I/R in-

jury in the local (intestine) and in the remote organ (lung), the

MPO activity was evaluated in tissue homogenates. Figure F11,

A and B, shows that 45 min of ischemia followed by a 4-h

reperfusion of the intestinal tissue caused significant neutro-

phil recruitment both in lung and in intestinal tissues. The

MPO activity reached its peak at 4 h after intestinal reper-

fusion and returned almost to its basal levels after 24 h. Fur-

thermore, intestinal I/R damage induces a significant increase

of LDH activity in serum (Fig. 1C) and a decrease of LDH in

FIG. 3. Cytokines levels in serum from mice upon intestinal I/R. Groups of mice were submitted to 45 min of intestinal ischemia followed by 4 h ofreperfusion. Sham-operated mice were used as controls. Basal values were obtained from naive mice. Cytokine levels were determined in serum usingmultiplex analysis IL-1! (A), IL-6 (B), KC (C), TNF-! (D), G-CSF (E), and MCP-1 (F). Data represent mean T SEM from 2 experiments from 5 to 7 animals.*P G 0.05.



the gut homogenates (Fig. 1D) as evidence of tissue injury

caused by intestinal I/R.

The neutrophilic inflammation was confirmed by micro-

scopic investigations of the lungs (Fig. 1F). We observe

neutrophil recruitment in the interstitial space and capillaries

of the lung upon intestinal I/R injury (Fig. 1H). There are also

signs of edema and inflammatory cells in the perivascular

space in the small vessels (Fig. 1F).

Cytokine profile in distinct compartments afterintestinal I/R injury

To identify major changes in the pattern of inflammatory

mediators expressed upon intestinal I/R injury, we evaluated

the expression of 22 cytokines and chemokines using a Linco

Multiplex kit in the homogenates of ileum and lung as well as

in the serum 4 h after intestinal reperfusion.

In the ileum homogenate, the levels of IL-1!, IL-6, KC,G-CSF, monocyte chemotactic protein 1 (MCP-1AQ3 ), and TNF-!are increased (F2 Fig. 2). The levels of the others cytokines and

chemokines are summarized in supplemental Table 1 (see Sup-

plemental Digital Content 1, http://links.lww.com/SHK/A36).

In addition, macrophage inflammatory protein 1! is upregu-

lated upon intestinal I/R injury in the ileum, whereas the other

mediators such as IL-2, IL-4, IL-12, IL-15, IL-17, IFN-+, andregulated upon activation, normal T cell expressed and secreted

were below the detection levels.

In the serum, a similar profile of that in lung homogenates is

observed, where the levels of IL-1!, IL-6, G-CSF, MCP-1, and

KC were increased in the intestinal I/R group as compared with

sham group ( F3Fig. 3 and Table, Supplemental Digital Content 2,

http://links.lww.com/SHK/A37). It is important to note that

there was no significant increase of circulating TNF-! upon

intestinal I/R injury.

In the lung, only IL-1!, IL-6, G-CSF, MCP-1, and KC were

upregulated upon intestinal I/R injury compared with the sham-

operated group, TNF-! concentration was increased less than a

factor of 2 upon intestinal I/R injury ( F4Fig. 4 and Table, Sup-

plemental Digital Content 3, http://links.lww.com/SHK/A38).

Therefore, upon intestinal I/R, the TNF-! levels in intestine

were slightly increased, whereas no increase was observed

in serum. However other cytokines and chemokines must be

important in this model.

FIG. 4. Cytokines levels in lung from mice upon intestinal I/R. Groups of mice were submitted to 45 min of intestinal ischemia followed by 4 h ofreperfusion. Sham-operated mice were used as controls. Basal values were obtained from naive mice. Cytokine levels were determined in lung homogenateusing multiplex analysis: IL-1! (A), IL-6 (B), KC (C), TNF-! (D). Data represent mean T SEM from 2 experiments from 5 to 7 animals. *P G 0.05.

AQ4



Studies of TNF-! involvement in I/R-induced lung andgut inflammation

Although TNF-! levels were not detectable in serum and

lung, we decided to investigate the potential role of this cyto-

kine in the inflammatory process induced by intestinal I/R

injury by the use of neutralizing TNF-! mAb. The admin-

istration of a neutralizing TNF-! mAb 30 min before intes-

tinal ischemia however did not reduce neutrophil recruitment

as assessed by MPO activity in lung and intestinal tissues

( F5Fig. 5, A and B). Moreover, the serum levels of cytokines

and chemokines in mice subjected to intestinal I/R were

not influenced by the administration of the TNF-! mAb

(Fig. 5, CYH, and Table, Supplemental Digital Content 4,

http://links.lww.com/SHK/A39).

To exclude a role of TNF-! in lung neutrophilic inflam-

mation in this model of intestinal I/R, we further used TNF

geneYdeficient mice. However, the MPO activity in the lung

from wild-type and TNF-deficient mice did not differ when

FIG. 5. Effects of the neutralization of soluble TNF-! in lung (A) and intestine (B) MPO activity and in the cytokine profile of mice subjected tointestinal I/R. Mice were treated with 20 mg/kg of antiYTNF-! mAb. Thirty minutes after the treatment, mice were subjected to 45 min of intestinal ischemiafollowed by 4 h of reperfusion. Mice treated with PBS were used as controls. Cytokine levels were determined in serum using multiplex analysis: IL-1! (C), IL-6(D), KC (E), TNF-! (F), G-CSF (G), and MCP-1 (H). Data represent mean T SEM from 2 experiments from 5 to 7 animals. *P G 0.05.



submitted to intestinal I/R (F6 Fig. 6A). Furthermore, neutro-

philic inflammation was investigated in mice deficient for

both TNF receptors 1 and 2 (TNFR1/R2 KO). In agreement

with the data from both TNF-! mAb treatment and TNF KO

experiments, MPO activity remains unchanged in the lung

tissues of mice undergoing intestinal I/R injury in the absence

of TNFR1/R2 signaling (Fig. 6B). Therefore, for this model of

intestinal I/R, we can safely conclude that lung inflammation

is TNF-! independent. We also analyzed mice at 8 h after

intestinal I/R injury and did not find any consistent reduction

of the global inflammatory response in TNF or TNFR1/R2

KO mice (data not shown).

TLRs mediate the lung inflammation induced byintestinal I/R

Intestinal ischemia causes substantial damage in the intes-

tinal wall as shown before with leak of intestinal microbes and

microbial products that activate the innate immunity through

TLR ligation. Therefore, we asked whether ablation of the

two major TLRs recognizing intestinal products, TLR2 and

TLR4, would alter the inflammatory response after intestinal

I/R injury. Indeed, pulmonary MPO activity was significantly

decreased in TLR2/4 doubleYdeficient mice when compared

with their wild-type controls (F7 Fig. 7A). We also investigated

the expression of TLR2 and TLR4 in lung tissue in both naive

and intestinal I/R mice and observed that TLR2 mRNA levels

are significantly increased in wild-type mice subjected to

intestinal I/R, whereas no significant upregulation of mRNA

of TLR4 was detected (Fig. 7, B and C), which does not ex-

clude signaling in response to intestinal I/R damage. There-

fore, the data suggest that microbial products augment at least

TLR2 expression and signal through TLR2/4 to induce the

inflammatory response.

DISCUSSION

This study in the murine model of intestinal I/R injury pro-

vides comprehensive data on a wide range of cytokines and

chemokines produced by the intestines and lung tissue and

released in the serum. Despite a pivotal role in many acute

inflammatory responses (19), we confirm our hypothesis that

TNF-! plays no role in remote inflammation induced by in-

testinal I/R injury. Neither the neutralization of soluble TNF-!nor the absence of TNF-! or its receptors prevented lung in-

flammation upon intestinal I/R injury, supporting the view that

TNF-! is not essential for the development of neutrophilic

inflammation nor did it alter the pattern of other proinflamma-

tory cytokines and chemokines in this model of intestinal I/R.

FIG. 6. Lung MPO activity in TNF KO and TNFR1/R2 KO micesubjected to intestinal I/R. Groups of TNF KO (A) and TNFR1/R2 KO(B) mice and wild-type mice were submitted to 45 min of intestinal ischemiafollowed by 4 h of reperfusion. Sham-operated mice were used as controls.Data represent mean T SEM from 2 experiments from 5 to 7 animals.*P G 0.05.

FIG. 7. Lung MPO activity in TLR2/4 KO mice subjected to intestinalI/R and expression of TLR2 and TLR4 mRNA in lung homogenates.Groups of mice C57Bl/6 or TLR2/4 were submitted to 45 min of intestinalischemia followed by 4 h of reperfusion. Basal values were obtained fromnaive mice. Expression of TLR2 (B) and TLR4 (C) mRNA were determinedby real-time PCR. *P G 0.05 relative to naive group.

AQ5



However, we report that TLRs, notably TLR2/4, play a critical

role for the induction of neutrophil recruitment into the lung as

observed by increased lung MPO activity.

The intestinal I/R injury procedure as performed in this

study is a simple and reproducible model of local and sys-

temic inflammation with significant repercussions to lung and

gut homeostasis (17, 20, 21). We used 45 min of intestinal

ischemia followed by 4 h or 24 h of intestinal reperfusion, a

condition considered as a mild model of injury with an in-

flammatory response characterized by neutrophil recruitment

to gut and lung tissues that peaks at 4 h after with no sig-

nificant mortality.

Although experimental data have been reported on the role

of individual cytokines in lung injury after intestinal I/R and

other forms of injury, no report so far exists on a broader panel

of cytokines/chemokines. Here, we investigated a large number

of cytokines and chemokines in ileum, lung, and serum of mice

subjected to intestinal I/R injury using a multiplex platform

(22). Such a comprehensive description of the changes of

cytokines/chemokines in three different compartments might

provide a better understanding of the role of the systemic

inflammation induced by intestinal I/R injury on the multiple

organ disease. We demonstrate here increased levels of IL-1!,IL-6, G-CSF, KC, MCP-1, macrophage inflammatory protein

1!, and TNF-! in the intestines. These factors are clearly

relevant to mediate acute inflammation and intestinal tissue

injury. Therefore, we extend previous data showing upregula-

tion of platelet-activating factorAQ6 (23), IL-1" (24), IL-6, and

TNF-! (25) in the intestine. Furthermore, our data support the

notion that cytokines/chemokines produced upon intestinal

injury drive remote inflammation in the lung. IL-1!, IL-6, G-CSF, and KC were upregulated in lung and in serum after

intestinal I/R injury. The gut, the initial site of ischemic injury

with highest levels of cytokines/chemokines, may spill its in-

flammatory mediators causing remote inflammation as sug-

gested for hemorrhagic shock by Deitch (26) and previously for

intestinal I/R injury (17, 27).

Of great interest was the observation that KC level in lung

homogenates from intestinal I/R mice were greater than

400 pg/mL upon intestinal I/R injury as compared with levels

of 100 pg/mL in the sham-operated group. This elevated level

of KC in lung tissue is even higher than that found in serum,

suggesting that this chemokine is produced locally in the lung

after intestinal I/R. In fact, Wiedermann et al. (28) reportedthat IL-8 (the human equivalent for KC) is increased in the

bronchoalveolar lavage of patients with ARDS, and that this

chemokine may be considered the main factor modulating the

influx of neutrophils in the early stage of the disease.

Unexpectedly, there was no change in TNF-! levels in both

serum and lung homogenates. Serum samples were taken also

1 h after the beginning of reperfusion to exclude a possible

early peak of TNF-!, but no increased serum levels were

observed (8.0 T 1.5 for naive mice, 12.9 T 1.3 for sham mice,

and 10.2 T 2.2 for mice upon intestinal I/R). Therefore, TNF-!seems to be unrelated to induction of lung inflammation in

our model of intestinal I/R. Interestingly, Davidson et al. (29)

did not implicate TNF-! in endothelial cell injury caused

by hemorrhagic shock. Several studies suggested that TNF-!

along with other cytokines may be important mediators of

acute lung injury (30, 31). However, elevated TNF-! levels in

serum and bronchoalveolar lavage fluid have not been con-

sistently correlated with clinical outcomes in patients at risk

of or with established acute lung injury (7).

Another important point in the discussion of the involve-

ment of TNF-! in intestinal I/R is the different roles that

this cytokine may play in the inflammatory process. Chen

et al. (11) showed that the activation of TLR4 is capable of

limiting the murine intestinal injury upon intestinal I/R, and

that this protective effect is mediated by TNF-!. In addition,

Teoh et al. (32) showed that a low dose of TNF-! admin-

istered before liver ischemia may have a protective effect.

The discrepant results from our data may be explained by a

different protocol used for intestinal I/R in the former study.

Noteworthy, these studies did not preclude a pivotal role of

TNF-! in acute inflammation, but suggest that TNF-! could

have a more complex regulatory role. Our experimental data

demonstrated that TNF-! does not participate in the modu-

lation of some of the hallmarks of systemic inflammation

caused by intestinal I/R injury. First, neutralization of TNF-!with antibody treatment did not diminish neutrophilic inflam-

mation and cytokine or chemokine levels in lung and gut upon

intestinal I/R injury. Second, the lack of TNF-! as inves-

tigated in TNF KO AQ7and in TNFR1/R2 double KO mice sub-

mitted to intestinal I/R injury did not abrogate inflammation.

These data represent strong evidence that TNF-! is not asso-

ciated with acute inflammation of the lung.

In contrast to the lack of involvement of TNF-!, TLR2 and

TLR4 showed prominent roles in neutrophil recruitment after

intestinal I/R injury. In fact, when we analyze lung homoge-

nates, there was a significant reduction in MPO activity of

TLR2/4 KO mice submitted to intestinal I/R injury when

compared with their C57Bl/6 controls. Furthermore, quantifi-

cation of mRNA by real-time PCR shows a small but sig-

nificant increase of TLR2 mRNA expression in lung tissue

upon intestinal I/R injury. These data demonstrate that TLR2

and TLR4 are important for neutrophil recruitment and

suggest a possible bacterial translocation as the source of in-

flammation induced by intestinal I/R. However, in a rat model

of intestinal I/R injury inflammation, inflammatory mediators

preceded increased serum endotoxin levels (33). Furthermore,

induction of oral tolerance to LPS or treatment with poly-

myxin B did not alter the inflammation and pulmonary edema

induced by intestinal I/R injury (33). Clearly, more work is

needed to understand the ligand-activating TLR2/4, which

play a critical role in pathophysiology (34). Toll-like receptors

may also recognize endogenous ligands such as heat shock

proteins and extracellular matrix components that could act as

Bdanger signals[ (14, 34, 35).

In conclusion, intestinal I/R injury induced the upregulation

of IL-6, G-CSF, and KC expression in local and remote organs,

which seems to play a role in pulmonary neutrophil recruitment

and inflammation, whereas TNF-! is not important for this

response. Importantly, TLR2/4 signaling caused by microbial

products accessing the body upon intestinal injury is critical to

initiate the inflammatory response and remote inflammation in

the lung.



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AUTHOR QUERIES

AUTHOR PLEASE ANSWER ALL QUERIES

AQ1 = WTOW was changed to WWITHW in the article title.

AQ2 = Please provide the expanded form of KC at first mention.

AQ3 = Please check if the expanded forms of MCP-1, MIP-1-alpha, and RANTES are correct.

AQ4 = Please provide significance of part E and F of figure 4 which were not mentioned in thecaption.

AQ5 = Please provide significance of part A of figure 7 which was not mentioned in the caption.

AQ6 = Please check if the expanded form of PAF is correct.

AQ7 = WTNF-W was changed to WTNF KO.W Please check if appropriate.

END OF AUTHOR QUERIES


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TUMOR NECROSIS FACTOR IS NOT ASSOCIATED WITH INTESTINAL ISCHEMIA/REPERFUSION-INDUCED LUNG...

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