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Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 2010 Oct;162(1):169–177. doi: 10.1111/j.1365-2249.2010.04225.x

Endotoxin-induced lung injury in α-galactosylceramide-sensitized mice is caused by failure of interleukin-4 production in lung natural killer T cells

J Dagvadorj 1, G Tumurkhuu 1, Y Naiki 1, A S M Noman 1, I Iftakhar-E-Khuda 1, B Badamtseren 1, T Komatsu 1, N Koide 1, T Yoshida 1, T Yokochi 1
PMCID: PMC2990943  PMID: 20659123

Abstract

Administration of bacterial lipopolysaccharide (LPS) known as endotoxin into α-galactosylceramide (α-GalCer)-sensitized mice causes severe lung lesions but few hepatic lesions in lethal shock, and interferon (IFN)-γ is suggested to play a pivotal role in preparation of the lung lesions. In order to clarify the mechanism of how α-GalCer sensitization causes lung lesions exclusively in mice, we examined the differential responsiveness of lungs and livers to α-GalCer sensitization. Although lung and liver natural killer T (NK T) cells both produced IFN-γ in response to α-GalCer, IFN-γ signalling was triggered only in the lungs of α-GalCer-sensitized mice. Lung NK T cells did not produce interleukin (IL)-4 in response to α-GalCer and it did not induce the expression of suppressor of cytokine signalling 1 (SOCS1) in the lungs. Conversely, IL-4 produced by liver NK T cells led to the expression of SOCS1 in the livers of the mice. Neutralization of IL-4 reduced SOCS1 expression in the livers and exacerbated LPS-induced hepatic lesions. IL-10 was produced by liver NK T cells but not lung NK T cells. However, IL-10 was produced constitutively by alveolar epithelial cells in normal lung. Lung NK T cells and liver NK T cells might express CD8 and CD4, respectively. Based on the fact that IL-4 inhibited IFN-γ signalling in the livers of α-GalCer-sensitized mice via SOCS1 expression and signal transducer and activator of transcription 1 (STAT-1) activation, no inhibition of the IFN-γ signalling in the lungs caused LPS-induced lung lesions in α-GalCer-sensitized mice. The detailed mechanism of development of the lung lesions in α-GalCer-sensitized mice is discussed.

Keywords: interferon (IFN)-γ, interleukin (IL)-4, lipopolysaccharide, NK T cell, α-galactosylceramide

Introduction

Bacterial lipopolysaccharide (LPS) as endotoxin stimulates the release of proinflammatory mediators and causes the systemic inflammatory response syndrome, endotoxic shock, disseminated intravascular coagulation and finally multi-organ failure [1,2]. LPS-mediated lethality has been characterized by a number of laboratory models. However, they are accompanied by hepatic injury and none of them represents respiratory failure, a typical manifestation in severe septic patients. Recently, we have established a new experimental model of endotoxic shock using α-galactosylceramide (α-GalCer)-sensitized mice [3]. The LPS-mediated lethal shock model using α-GalCer is accompanied by severe lung lesions with marked infiltration of inflammatory cells and massive cell death. Conversely, hepatic lesions were focal and slight. The experimental model is consistent with clinical features of severe septic shock in patients. LPS-mediated lethal shock with α-GalCer sensitization is useful for the understanding of clinical sepsis and septic shock [3].

LPS-mediated lethal shock using α-GalCer sensitization does not occur in Vα14-positive natural killer T (NK T) cell-deficient mice [3]. Based on analysis using anti-interferon (IFN)-γ or tumour necrosis factor (TNF)-α neutralizing antibody, IFN-γ and TNF-α play respective roles on the preparation and development of the lung lesions. The successive study has reported that IFN-γ produced by α-GalCer-activated NK T cells induces the expression of vascular cell adhesion molecule (VCAM)-1 in the lungs of α-GalCer-sensitized mice and that very late antigen (VLA)-4-positive cells as the counterpart of VCAM-1 accumulate into the lungs [4]. The subsequent exposure of VLA-4-positive cells to LPS results in the release of excessive TNF-α. Finally, an excess of TNF-α leads to the elevation of pulmonary permeability and cell death [4]. This is the putative mechanism of acute lung injury in LPS-mediated lethal shock using α-GalCer sensitization. Conversely, IFN-γ produced by α-GalCer-stimulated NK T cells induces no VCAM-1 expression in the livers of α-GalCer-sensitized mice and causes few hepatic lesions [4]. Therefore, it is of particular interest to characterize how IFN-γ produced by α-GalCer-stimulated NK T cells sensitizes the lungs but not the livers for the development of tissue lesions.

α-GalCer has been identified as a glycolipid ligand, which stimulates a special group of NK T cells [5–7]. NK T cells rapidly produce IFN-γ, interleukin (IL)-4 and IL-10 in response to α-GalCer [8]. The differential response between the lung and the liver to α-GalCer might be dependent upon the functional difference in α-GalCer-stimulated NK T cells between two organs. In the present work we studied how α-GalCer sensitization selectively caused lung lesions in LPS-mediated lethal shock via activation of NK T cells. Here, we report that no IL-4 production by lung NK T cells may lead to the selective development of lung lesions in LPS-induced lethal shock using α-GalCer-sensitized mice.

Materials and methods

Mice

BALB/c mice, approximately 7 weeks of age, were supplied by Japan SLC (Hamamatsu, Japan). All animal experiments were approved by the Animal Care Committee of Aichi Medical University and carried out under the guide for care and use of laboratory animals.

Reagents

α-GalCer and monensin were obtained from Funakoshi (Tokyo, Japan), and LPS from Escherichia coli O55:B5 was from Sigma Chemicals (St Louis, MO, USA). Anti-IL-10 and anti-IL-4 neutralizating monoclonal antibodies (mAbs) and an enzyme-linked immunosorbent assay (ELISA) kit for mouse IFN-γ were obtained from R&D Systems (Minneapolis, MN, USA). Percoll and collagenase were purchased from GE Healthcare Bio-sciences AB (Uppsala, Sweden) and Wako (Osaka, Japan), respectively.

Reverse transcription–polymerase chain reaction (RT–PCR) and real-time PCR

The total RNA was isolated from tissues using a RNeasy minikit (Qiagen Sciences, Gaithersburg, MD, USA) in accordance with the manufacturer's protocol. Total RNA was reverse-transcribed to cDNA using a RT system with random hexamers (Toyobo, Tokyo, Japan). Expression of mRNA was analysed with StepOne real-time PCR, according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). The reaction mixture consists of SYBR green PCR master mix (Toyobo) and sequence-specific primers: 18S rRNA sense, 5′-TGACTCAACACGGGAAACC-3′, anti-sense, 5′-TCGCTCCACCAACTAGAAC-3′; IFN-γ sense, 5′-GCTTTAACAGCAGGCCAGAC-3′, anti-sense, 5′-GGAAGCACCAGGTGTCAAGT-3′; IL-10 sense, 5′-CCAGGGAGATCCTTTGATGA-3′ anti-sense, 5′-AACTGGCCACAGTTTTCAGG-3′; IL-4 sense, 5′-GCAACGAAGAACACCACAGA-3′, anti-sense, 5′-GCATGGAGTTTTCCCATGTT-3′.

Immunoblotting

Lungs and livers were homogenized with a nuclear extract kit (Active Motif, Carlsbad, CA, USA) and the insoluble fraction was removed by centrifugation. The protein concentration was determined by the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL, USA). Each sample containing 35 µg of protein was analysed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. The membranes were treated with 5% skimmed milk for 3 h and incubated with a series of antibodies to phosphorylated signal transducer and activator of transcription 1 (STAT-1), phosphorylated STAT-3 (Cell Signalling, Danvers, MA, USA), cytokine signalling 3 (SOCS-3) (UpState Biotechnology, Hamburg, Germany), SOCS-1 (AnaSpec, San Jose, CA, USA) and IFN regulatory factor (IRF)-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibodies were used at a 1:500 dilution unless stated otherwise. The immune complexes were detected with a 1:1000 dilution of horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody (eBioscience, San Diego, CA, USA) and the bands were visualized with a chemiluminescent reagent (Pierce, Rockford, IL, USA). The chemiluminescence was analysed by a light capture system (AE6955; Atto Corporation, Tokyo, Japan) with CS analyser.

Preparation of lung and liver mononuclear cells

Mononuclear cells from lungs and livers were prepared as described elsewhere [4,9]. In brief, lungs were perfused with phosphate-buffered saline (PBS) and sliced into small pieces. The lobes were incubated with RPMI-1640 containing 1% fetal calf serum, DNase (1 mg/ml), collagenase (1 mg/ml) and monensin (3 µM) for 30 min. The tissue suspension was aspirated gently and expelled from a syringe through a 22-g needle to assist tissue disruption every 5 min. Livers were minced, passed through a mesh and suspended in RPMI-1640 containing monensin (3 µM). After washing, the mononuclear cells were separated by centrifugation on discontinuous Percoll gradients (45%/67%).

Laser flow cytometry and sorting

Cells were suspended in 100 µl PBS containing 1% bovine serum albumin and incubated with mouse Fc receptor blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) on ice for 15 min. The cells were stained with allophycocyanin (APC)-conjugated anti-mouse CD3ε antibody, fluorescein isothiocyanate (FITC)-conjugated anti-pan NK (CD49b) DX5 antibody, phycoerythrin (PE)-conjugated anti-mouse CD4 antibody or PE/Cy7-conjugated anti-mouse CD8 antibody (BioLegend, San Diego, CA, USA). Isotype-matched irrelevant antibodies were used as the control antibody. The cells were treated with fixation and permeabilization buffer (eBioscience, San Diego, CA, USA) and stained with PE-conjugated antibodies to mouse IFN-γ, IL-10 and IL-4 (BioLegend) for detection of intracellular expression. The laser flow cytometric analysis and sorting were performed by fluorescence activated cell sorter (FACS) Cando II (BD Biosciences, Mountain View, CA, USA).

Immunohistochemical staining

Lung or liver paraffin sections were deparaffinized, blocked with methanol containing 3% hydrogen peroxide and control serum, and then incubated with anti-IL-10 antibody (Santa Cruz Biotechnology) at appropriate dilutions overnight. The immune complexes were detected with horseradish peroxidase-conjugated EnVision + System labelled polymer (DakoCytomation, Carpinteria, CA, USA) and 3,3′-diaminobenzidene tetrachloride (Sigma). The sections were counterstained with methylgreen. Photographs were taken by the Fujix digital camera HC-2500 under an Olympus BX50 microscope.

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) specific for fragmented DNA

Mice were injected intravenously (i.v.) with α-GalCer (1 µg) and 12 h later LPS (5 µg) was injected i.v. into the mice. Eight mice were used in each experimental group. Lungs and livers were removed 5 h after LPS injection, unless stated otherwise. They were fixed with 10 % formalin and cut serially into 4–6 µm sections. For detection of fragmented DNA, the paraffin sections were deparaffinized and stained with the TUNEL method as described previously [10,11].

Statistical analysis

Statistical analysis was performed using Student's t-test, with P < 0·01 considered to indicate a significant difference. Experimental results are expressed as the mean value of triplicates with standard deviation (s.d.) in at least three independent experiments.

Results

α-GalCer induces IFN-γ production in both lung and liver NK T cells

NK T cells are known to produce IFN-γ in response to α-GalCer [6,12]. The IFN-γ productivity of lung and liver NK T cells in response to α-GalCer was examined (Fig. 1). Mice were injected i.v. with α-GalCer (1 µg) and mononuclear cells were isolated from lungs and livers of the mice 6 h after α-GalCer treatment based on the kinetic of IFN-γ in the previous study [4]. NK T cells were identified as pan-NK (CD49b) and CD3ε-positive cells and the intracellular IFN-γ expression was determined by FACS analysis (Fig. 1a). Intracellular IFN-γ was expressed markedly in both NK T cells from lungs and livers of α-GalCer-injected mice. There was no significant difference in intracellular IFN-γ expression between lung and liver NK T cells. The expression of IFN-γ protein and mRNA in the lungs and livers was also determined by ELISA and real-time PCR, respectively (Fig. 1b). α-GalCer induced the expression of IFN-γ protein and mRNA significantly in the lungs and livers.

Fig. 1.

Fig. 1

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α-Galactosylceramide (α-GalCer)-induced interferon (IFN)-γ production in lung and liver natural killer T (NK T) cells. (a) The intracellular IFN-γ production in lung and liver NK T cells was analysed 6 h after α-GalCer treatment with a fluorescence activated cell sorter. (b) The levels of IFN-γ protein in homogenates and IFN-γ mRNA in NK T cells were determined with enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. The mRNA expression was determined 6 h after α-GalCer treatment and normalized using the housekeeping gene 18S rRNA. The fold increase was calculated based on the value of untreated control. (c) The expression of phosphorylated signal transducer and activator of transcription 1 (pSTAT-1) and IFN regulatory factor (IRF)-1 in lung and liver extracts was analysed by immunoblotting. Lungs and livers were removed at the indicated time after α-GalCer treatment. A typical result in three independent experiments is shown.

IFN-γ signalling is triggered in lungs but not livers of α-GalCer-sensitized mice

Because IFN-γ was produced in both lungs and livers of α-GalCer-injected mice, we examined the activation of IFN-γ signalling in the lungs and livers. The lungs and livers were removed 4, 8 or 12 h after the injection of α-GalCer and their homogenates were analysed with immunoblotting using anti-phosphorylated STAT-1 or anti-IRF-1 antibody (Fig. 1c). The expression of phosphorylated STAT-1 was augmented significantly in lungs and livers 4 h after the α-GalCer injection. The augmented expression of phosphorylated STAT-1 in the lungs continued for up to 12 h, whereas that in the livers clearly declined at 8 h. IRF-1 expression was augmented significantly in lungs but not in livers. Although α-GalCer induced IFN-γ production in both lung and liver NK T cells, the successive IFN-γ signalling was triggered exclusively in lungs but not livers.

Liver NK T cells but not lung NK T cells produce IL-10 in response to α-GalCer

In the preceding section, IFN-γ signalling was triggered in lungs but not livers in α-GalCer-injected mice. Therefore, a possibility was raised that IFN-γ signalling in livers might be inhibited by anti-inflammatory cytokine, such as IL-10 [13,14]. IFN-γ production and signalling occurred 4 h after α-GalCer stimulation (Fig. 1). Cytokines affecting IFN-γ production and signalling must be produced within 4 h. Therefore, further characterization of the action of IL-10 and IL-4 was examined 2 h after α-GalCer stimulation. First, we examined the production of IL-10 in lungs and livers from α-GalCer-sensitized mice. Mice were injected i.v. with α-GalCer (1 µg), and lungs and livers were removed 2 h after the injection. NK T cells were identified with anti-pan NK and anti-CD3ε antibodies and intracellular IL-10 expression was determined with laser flow cytometric analysis (Fig. 2). Laser flow cytometric analysis demonstrated that liver NK T cells definitely expressed intracellular IL-10, whereas lung NK T cells did not (Fig. 2a). Further, we examined the expression of IL-10 mRNA in NK T cells. As shown in Fig. 2b, pan NK+ and CD3+ cells were sorted as NK T cell population from total lymphocytes of lungs and livers and IL-10 mRNA expression was determined by real-time PCR. IL-10 mRNA was expressed clearly in liver NK T cells but not lung NK T cells. The impaired IFN-γ signalling in livers of α-GalCer-sensitized mice might be responsible for IL-10 production by liver NK T cells.

Fig. 2.

Fig. 2

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α-Galactosylceramide (α-GalCer)-induced interleukin (IL)-10 production in lung and liver natural killer T (NK T) cells. (a) The intracellular IL-10 production in lung and liver NK T cells was analysed 2 h after α-GalCer treatment with fluorescence activated cell sorter. (b) The IL-10 mRNA expression in NK T cells was determined at 2 h by real-time polymerase chain reaction. The mRNA expression was normalized using the housekeeping gene 18S rRNA and the fold increase was calculated based on the value of untreated control. *P < 0·01 versus lung.

IL-10 is expressed constitutively in normal lungs

Based on the finding that IL-10 is expressed in liver NK T cells but not lung NK T cells, IL-10-induced STAT-3 and SOCS-3 activation was examined in livers and lungs of α-GalCer-sensitized mice. Mice were injected i.v. with α-GalCer (1 µg) and then lungs and livers were removed from mice 4, 8 or 12 h after the α-GalCer injection (Fig. 3a). In livers of α-GalCer-sensitized mice, STAT-3 phosphorylation was clearly detected at 4 h and then declined gradually. SOCS-3 expression was augmented significantly 8 h after α-GalCer injection and increased further up to 12 h. Surprisingly, SOCS-3 expression and STAT-3 phosphorylation were also detected in the lungs even before α-GalCer injection, indicating constitutive IL-10 expression in the lungs. Phosphorylation of STAT-3 was augmented 4 h after α-GalCer treatment and decreased gradually until 12 h. Because of spontaneous expression of SOCS-3 and phosphorylation STAT-3, we examined immunohistochemically constitutive IL-10 expression in lungs of normal control mice (Fig. 3b). The sections of lungs and livers from normal mice were stained with anti-IL-10 antibody or isotype control antibody. Lung alveolar epithelial cells were stained positively with anti-IL-10 antibody. There was no significant difference in IL-10 expression in the lung extracts between α-GalCer-treated and untreated mice (data not shown). Conversely, there was no positive IL-10 staining in normal liver sections. Thus, IL-10 was expressed on alveolar epithelial cells in normal lungs.

Fig. 3.

Fig. 3

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Constitutive interleukin (IL)-10 expression in lungs. (a) Lungs and livers were removed at the indicated time after α-galactosylceramide (α-GalCer) treatment. The expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT-3) and cytokine signalling 3 (SOCS-3) in lung and liver extracts was determined by immunoblotting. A typical result in three independent experiments is shown. (b) The IL-10 expression in normal lungs and livers was determined by immunohistochemical staining using an anti-IL-10 antibody. A typical result in three independent experiments is shown. (c) Effect of anti-IL-10 monoclonal antibody on α-GalCer-induced interferon (IFN)-γ production was determined with enzyme-linked immunosorbent assay. Anti-IL-10 antibody was injected intravenously 1 h before α-GalCer treatment and the level of IFN-γ was determined 12 h after α-GalCer treatment.

Next, the effect of anti-IL-10 neutralizing antibody on IFN-γ production in α-GalCer-sensitized mice was examined (Fig. 3c). Anti-IL-10 neutralizing antibody or isotype control pretreated mice were injected i.v. with α-GalCer, and lungs and livers were removed 12 h after α-GalCer injection. The level of IFN-γ expression in lung or liver homogenates was determined by ELISA. Anti-IL-10 antibody enhanced IFN-γ production significantly in both lungs and livers, suggesting that IL-10 inhibited IFN-γ production in α-GalCer sensitized mice. However, the differential response in IFN-γ signalling between lung and liver could not be elucidated by production of anti-inflammatory IL-10.

NK T cells in liver but not lung produce IL-4 in response to α-GalCer

IL-4 as well as IL-10 inhibits IFN-γ signalling [15] and NK T cells produce IL-4 in response to α-GalCer [16]. The involvement of IL-4 on the differential response of lungs and livers in IFN-γ signalling was examined (Fig. 4). Mice were injected i.v. with α-GalCer (1 µg) and lungs and livers were removed 2 h after α-GalCer injection. NK T cells were gated by the positivity with anti-pan NK antibody and anti-CD3ε antibody, and intracellular IL-4 expression was analysed by laser flow cytometry (Fig. 4a). Liver NK T cells in α-GalCer-sensitized mice expressed intracellular IL-4 significantly, whereas lung NK T cells did not. The expression of IL-4 mRNA was determined with real-time PCR (Fig. 4b). Pan NK+ and CD3+ NK T cells were sorted by a FACS cell sorter using the antibodies. The IL-4 mRNA was expressed highly in liver NK T cells but not in lung NK T cells from α-GalCer-sensitized mice.

Fig. 4.

Fig. 4

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α-Galactosylceramide (α-GalCer)-induced interleukin (IL)-4 production in lung and liver natural killer T (NK T) cells. (a) The intracellular IL-4 expression in lung and liver NK T cells was analysed 2 h after α-GalCer treatment with a fluorescence activated cell sorter. (b) The IL-4 mRNA expression in NK T cells was determined by real-time polymerase chain reaction. *P < 0·01 versus lung. Lungs and livers were removed 2 h after α-GalCer treatment. The experimental result was normalized using the housekeeping gene 18S rRNA and the fold increase was calculated based on the value of untreated control. (c) Lungs and livers were removed at the indicated time after α-GalCer treatment. The expression of phosphorylated signal transducer and activator of transcription 6 (pSTAT-6) and cytokine signalling 1 (SOCS-1) in lung and liver extracts was determined with immunoblotting. A typical result in three independent experiments is shown.

Next, we examined the expression of SOCS-1 and phosphorylation of STAT-6, which are induced by IL-4, in the lungs and livers of α-GalCer-sensitized mice. Mice were injected i.v. with α-GalCer (1 µg) and lungs and livers were removed 4, 8 or 12 h after the α-GalCer injection. The SOCS-1 and phosphorylated STAT-6 expression was determined with immunoblotting (Fig. 4c). The SOCS-1 expression was detected clearly in liver homogenates 4–12 h after the α-GalCer injection. Conversely, slight SOCS-1 expression was detected in the lung homogenates at 8 and 12 h. The expression of phosphorylated STAT-6 was much more augmented in liver homogenates 4–12 h after α-GalCer injection than that in lung homogenates. IL-4 production in liver NK T cells was suggested to inhibit IFN-γ signalling via augmented SOCS-1 and phosphorylated STAT-6 expression in livers. Conversely, no IL-4 production in lung NK T cells might allow the triggering of IFN-γ signalling in the lungs. Thus, IL-4 might be a responsible effector for differential response of the liver and lung to IFN-γ.

Neutralization of IL-4 causes severe liver injury in α-GalCer-sensitized mice

The effect of IL-4 neutralization on the development of LPS-induced tissue lesions in lungs and livers of α-GalCer-sensitized mice was examined. α-GalCer (1 µg) were injected into mice pretreated with anti-IL-4 or IL-10 neutralizing antibody and 12 h later LPS (5 µg) was administered into the mice. Lungs and livers were removed 6 h after LPS injection and stained with TUNEL specific for fragmented DNA. Administration of LPS into α-GalCer-sensitized mice caused the appearance of a number of TUNEL-positive apoptotic cells in lungs but not in livers (Fig. 5a, top). Anti-IL-4 neutralizing antibody did not affect the TUNEL staining in the lungs. However, it caused the appearance of a number of positively stained apoptotic cells in the livers (Fig. 5a, middle). Anti-IL-10 antibody induced more apoptotic cells in lungs, suggesting the development of severe lung injury (Fig. 5a, bottom). Thus, IL-4 was suggested to play a critical role in preventing liver injury in α-GalCer-sensitized mice.

Fig. 5.

Fig. 5

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Effect of neutralization of interleukin (IL)-4 or IL-10 on lipopolysaccharide (LPS)-induced tissue lesions in α-galactosylceramide (α-GalCer)-sensitized mice. (a) Anti-IL-10 or anti-IL-4 monoclonal antibody was treated intravenously (i.v.) 1 h before α-GalCer (1 µg) sensitization and 12 h later lipopolysaccharide (LPS) (5 µg) was injected i.v. into the mice. Lungs and livers were removed 6 h after LPS treatment and apoptotic cells were stained with terminal deoxynucleotidyl transferase dUTP nick end labelling. (b) Lungs and livers were removed 8 h after α-GalCer treatment. The expression of phosphorylated signal transducer and activator of transcription 1 (pSTAT-1) and cytokine signalling 1 (SOCS-1) in lung and liver extract was analysed with immunoblotting. A typical result in three independent experiments is shown.

Next, we examined the effect of anti-IL-4 or IL-10 neutralizing antibody on the activation of STAT-1 and SOCS-1 in the lungs and livers of α-GalCer-sensitized mice. α-GalCer (1 µg) was injected i.v. into mice pretreated with anti-IL-4 or IL-10 antibody and lungs and livers were removed at 8 h. Phosphorylation of STAT-1 and expression of SOCS-1 were determined by immunoblotting with an antibody to phosphorylated STAT-1 or SOCS-1 (Fig. 5b). In livers, anti-IL-4 neutralizing antibody augmented phosphorylation of STAT-1 and reduced the expression of SOCS-1. Conversely, neither anti-IL-4 antibody nor anti-IL-10 antibody affected them in lungs. Therefore, IL-4 was suggested to prevent hepatic lesions in α-GalCer-sensitized mice through inhibiting IFN-γ signalling.

CD8+ NK T cells are present in lungs but not livers of normal mice

In the preceding section, liver NK T cells produced IL-4 and IFN-γ, whereas lung NK T cells produced only IFN-γ. Further, IL-4 was suggested to play a crucial role on the prevention of hepatic lesions in LPS-mediated lethal shock in α-GalCer-sensitized mice. Therefore, we examined the phenotypes of NK T cells between lung and liver with laser flow cytometry (Fig. 6). NK T cells were gated by positivity with anti-pan NK and anti-CD3ε antibody and the expression of CD4 and CD8 on the gated population was analysed. A significant number of CD8+ cells were present in lung NK T cells, whereas few such cells were detected in the livers. Conversely, liver NK T cells contained more CD4+ cells than lung NK T cells. In addition, the possibility was not excluded that pan NK-positive and CD3ε-positive cells gated contained other cells besides NK T cells.

Fig. 6.

Fig. 6

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Expression of CD4 and CD8 on lung and liver natural killer T (NK T) cells. NK T cells were gated by the positivity with anti-pan NK and anti-CD3 antibodies. The expression of CD4 and CD8 on the gated population was analysed with fluorescence activated cell sorter. A typical result in three independent experiments is shown.

Discussion

Recently, we have established a novel experimental model for clinical septic shock [3,4]. Although a number of experimental endotoxic shock models are accompanied by severe hepatic lesions, our model is characterized by severe lung lesions and few hepatic lesions, which is consistent with clinical septic shock [3,4]. The present study was carried out to elucidate the mechanism how LPS induces lung injury selectively in α-GalCer-sensitized mice. In the current study we demonstrate that liver NK T cells are able to produce IL-4 and prevent hepatic lesions through inhibiting IFN-γ signalling. Conversely, lung NK T cells are unable to produce IL-4, which inhibits IFN-γ signalling. Therefore, IFN-γ signalling is triggered exclusively in the lungs of α-GalCer-sensitized mice and results in the expression of adhesion molecules, such as VCAM-1, on vascular endothelial cells in the lungs [4]. Subsequently, VCAM-1 expression accumulates VLA-4-positive inflammatory cells in the lungs and those cells produce an excess of TNF-α in response to LPS [4]. The release of excessive TNF-α leads to the elevation of pulmonary permeability and massive cell death, followed by severe lung lesions [4]. This is the putative mechanism of development of lung lesions in LPS-mediated lethal shock using α-GalCer sensitization.

We demonstrate that IL-4 prevents the development of hepatic lesions via inhibition of IFN-γ signalling in α-GalCer-sensitized mice. IL-4 is reported to inhibit IFN-γ signalling through activating STAT-6 [16]. Further, IL-4 prevents IFN-γ signalling via augmented expression of SOCS-1, which regulates IFN-γ signalling negatively. SOCS-1 is known to inhibit IFN-γ signalling by binding directly to the IFN-γ receptor [17–19]. Moreover, mice lacking a functional SOCS-1 gene are hypersensitive to IFN-γ and have a number of characteristic lesions, such as hepatic necrosis, macrophage infiltration in several organs, multiple haematopoietic abnormalities and severe lymphopenia [20,21]. Thus, IL-4 produced by liver NK T cells is suggested to inhibit IFN-γ signalling via STAT-1 inactivation and SOCS-1 induction. It is also supported by the finding that anti-IL-4 neutralizing antibody enhances the STAT-1 activation in IFN-γ signalling and exacerbates hepatic lesions. Conversely, the failure of IL-4 production in lung NK T cells in response to α-GalCer is unable to inhibit IFN-γ signalling and causes severe lung lesions. Thus, IL-4 might be a main regulatory molecule in the development of LPS-mediated lung lesions in α-GalCer-sensitized mice.

Lung NK T cells produce IFN-γ exclusively, whereas liver NK T cells produce IL-4 and IL-10 as well as IFN-γ. Considering that IL-4 is the critical molecule for inhibition of LPS-induced hepatic lesion in α-GalCer-sensitized mice, the difference in the cytokine profile between lung and liver NK T cells might be responsible for the development of LPS-mediated lung injury. In fact, it has been reported that NK T cells are phenotypically, functionally and developmentally heterogeneous, and that three distinct subsets consisting of double-negative, CD4+ and CD8+ are distributed differentially in a tissue-specific fashion [22–24]. The majority of NK T cells in most tissues are either CD4+ or double-negative [25]. However, CD4- and CD8+ NK T cells are also present in all tissues except the thymus in mice [23,24,26,27]. In particular, CD4- and CD8+ NK T cells are dominant in the lung [23]. CD4+ or double-negative NK T cells produce IL-4 as well as IFN-γ, whereas CD8+ NK T cells produce only IFN-γ[26–28]. The possibility that lung NK T cells may express CD8+ and produce IFN-γ exclusively in response to α-GalCer is also supported by the present study. The difference in the cytokine profile between lung and liver NK T cells is suggested to cause the differential response between lungs and livers to IFN-γ and develop only lung lesions in α-GalCer-sensitized mice.

IL-10, an anti-inflammatory cytokine, is detected constitutively in normal lungs of human and mice, and is produced by immature dendritic cells, mononuclear phagocytic cells and alveolar epithelial cells [29,30]. The present study demonstrates that IL-10 inhibits IFN-γ production in the lungs and livers of α-GalCer-sensitized mice as anti-IL-10 neutralizing antibody enhances the IFN-γ production. However, IFN-γ signalling is triggered in the lung of α-GalCer-injected mice and leads to acute lung injury in response to LPS. Therefore, it is possible that IL-10 inhibits IFN-γ production but not IFN-γ signalling in the lungs of α-GalCer-sensitized mice. In fact, IL-10 is reported to induce SOCS-3 expression and inhibit the signalling of proinflammatory cytokines but not IFN-γ[31,32]. SOCS-1 is a more potent inhibitor of IFN-γ signalling than is SOCS-3 [33–36]. IL-10 is known to induce SOCS-1 in several cell lines, including Ba/F3 pro-B cells and M1 leukaemia cells [37]. However, there is no alteration in the SOCS1 expression in the livers of IL-10-neutralizing mice, although IFN-γ production was enhanced in the livers of α-GalCer-sensitized mice. It is consistent with the previous report that IL-10 indirectly suppresses the synthesis of IFN-γ[13,14,38]. Taken together, IL-10 may be involved in the inhibition but not the signalling of IFN-γ production. In addition, we do not necessarily exclude the involvement of IL-10 produced by alveolar epithelial cells in the prevention of lung injury via inhibition of IFN-γ production.

In the clinical setting, patients with severe septic shock are characterized by respiratory dysfunction, accompanied by lung lesions. The activation of lung NK T cells in pulmonary infections might lead to the production of IFN-γ without IL-4 capable of inhibiting IFN-γ signalling and inducing lung lesions. Moreover, more severe lung lesions would develop in the patients if constitutive IL-10 synthesis was impaired in alveolar epithelial cells. A series of complicated mechanisms might regulate the development of severe lung lesions in clinical septic shock. The involvement of NK T cells in the development of lung injury in human septic shock must await further characterization.

Acknowledgments

This work was supported by in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. We are grateful to K. Takahashi and A. Morikawa for technical assistance.

Disclosure

The authors declare no interests to disclosure.

References

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