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. 2008 Apr;152(1):182–191. doi: 10.1111/j.1365-2249.2008.03603.x

The mechanism of development of acute lung injury in lethal endotoxic shock using α-galactosylceramide sensitization

G Tumurkhuu 1, N Koide 1, J Dagvadorj 1, A Morikawa 1, F Hassan 1, S Islam 1, Y Naiki 1, I Mori 1, T Yoshida 1, T Yokochi 1
PMCID: PMC2384075  PMID: 18307519

Abstract

The mechanism underlying acute lung injury in lethal endotoxic shock induced by administration of lipopolysaccharide (LPS) into α-galactosylceramide (α-GalCer)-sensitized mice was studied. Sensitization with α-GalCer resulted in the increase of natural killer T (NK T) cells and the production of interferon (IFN)-γ in the lung. The IFN-γ that was produced induced expression of adhesion molecules, especially vascular cell adhesion molecule-1 (VCAM-1), on vascular endothelial cells in the lung. Anti-IFN-γ antibody inhibited significantly the VCAM-1 expression in α-GalCer-sensitized mice. Very late activating antigen-4-positive cells, as the counterpart of VCAM-1, accumulated in the lung. Anti-VCAM-1 antibody prevented LPS-mediated lethal shock in α-GalCer-sensitized mice. The administration of LPS into α-GalCer-sensitized mice caused local production of excessive proinflammatory mediators, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and nitric oxide. LPS caused microvascular leakage of proteins and cells into bronchoalveolar lavage fluid. Taken together, sensitization with α-GalCer was suggested to induce the expression of VCAM-1 via IFN-γ produced by NK T cells and recruit a number of inflammatory cells into the lung. Further, LPS was suggested to lead to the production of excessive proinflammatory mediators, the elevation of pulmonary permeability and cell death. The putative mechanism of acute lung injury in LPS-mediated lethal shock using α-GalCer sensitization is discussed.

Keywords: acute lung injury, α-galactosylceramide, lipopolysaccharide, NK T cell, septic shock, VCAM-1

Introduction

Lipopolysaccharide (LPS), the component of the Gram-negative bacterial cell wall, stimulates the release of proinflammatory mediators from various cell types and causes the systemic inflammatory response syndrome, endotoxic shock, disseminated intravascular coagulation and multi-organ failure [1,2]. LPS-mediated lethality has been characterized by a number of laboratory models using sensitization with d-galactosamine [3], Propionibacterium parvum[4], bacille Calmette–Guérin [5] and cytosine–phosphate–guanine DNA [6]. Generalized Shwartzman reaction is a potentially lethal shock reaction, which can be induced by administration of LPS into LPS-primed animals [7]. None of them presents respiratory failure, a typical manifestation in severe septic patients. Therefore, there is a need for an animal model of sepsis characterized by respiratory failure severe enough to require mechanical ventilation.

Recently, we have established a new experimental model of endotoxic shock using α-galactosylceramide (α-GalCer) sensitization [8]. α-GalCer is a glycolipid which has been identified as a ligand stimulating a special group of natural killer T (NK T) cells [9,10]. The α-GalCer-sensitized mice are killed by administration of a small amount of LPS. LPS-mediated lethal shock is accompanied by severe lung lesions with marked infiltration of inflammatory cells and massive cell death. The model using α-GalCer sensitization clearly presents acute lung injury and respiratory failure, and is consistent with the clinical features of severe septic shock. Thus, the model is useful for the characterization of acute lung injury in severe sepsis and septic shock in humans [8]. Further, interferon (IFN)-γ and tumour necrosis factor (TNF)-α is reported to play a critical role in the preparation and execution of the lethal shock respectively [8]. In the present work, we studied the mechanism of development of acute lung lesions in lethal endotoxic shock using α-GalCer sensitization in order to characterize clinically manifest respiratory failure in septic shock patients. Here, we report the respective roles of α-GalCer and LPS in sensitization and execution for lethal endotoxic shock.

Materials and methods

Mice

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

Reagents

α-galactosylceramide (KRN7000) was provided by Kirin Brewery Company (Gunma, Japan). LPS from Escherichia coli O55:B5 was obtained from Sigma Chemicals (St Louis, MO, USA). A monoclonal antibody to murine IFN-γ and vascular cell adhesion molecule-1 (VCAM-1) were obtained from R&D Systems (Minneapolis, MN, USA).

Induction of LPS-mediated lethal shock

Lipopolysaccharide-mediated lethal shock was developed based on the experimental system established in the previous study [8]. Briefly, mice were injected intravenously (i.v.) with α-GalCer (1 μg) for sensitization and then challenged i.v. with LPS (5 μg) 12 h after α-GalCer treatment unless stated otherwise. Approximately six mice were used in each experimental group.

Histology

The lungs were removed, fixed with 10% formalin and stained with haematoxylin and eosin. Histological changes were observed microscopically and photographs were taken using the Fujix digital camera HC-2500 under an Olympus BX50 microscope.

Immunohistochemical staining

Paraffin sections of lungs were deparaffinized and the endogenous peroxidase activity was blocked with methanol containing 3% hydrogen peroxide for 20 min at room temperature. The sections were incubated with phosphate-buffered saline (PBS) containing 1% normal control serum for 30 min to avoid non-specific staining and then incubated with antibodies to nitrotyrosine (Cell Signalling, Danvers, MA, USA) and inducible nitric oxide synthetase (iNOS) (BD Transduction Laboratories, San Diego, CA, USA) at appropriate dilutions for 60 min. For VCAM-1, E-selectin and intercellular cell adhesion molecule (ICAM-1) staining, frozen sections of lung were excised and the endogenous peroxidase activity was blocked. The sections were treated for 10 min with 1% bovine serum albumin solution and incubated with antibodies to VCAM-1, E-selectin (CD62E) and ICAM-1 (BD PharMingen, San Diego, CA, USA). 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. In three different tissue sections of each mouse, the expression of target proteins was assessed three times by a blinded investigator. Photographs were taken using the Fujix digital camera HC-2500 under an Olympus BX50 microscope.

Preparation of pulmonary intraparenchymal leucocytes and laser flow cytometry

The chest of the mouse was opened and the lung vascular bed was flushed by injecting chilled PBS (2 ml) into the right ventricle to avoid contamination of cells circulating in the blood. The aorta and inferior vena cava were cut and the lungs were removed. Pulmonary intraparenchymal leucocytes were prepared from the lungs as described elsewhere [11]. Briefly, the lungs were excised, sliced into small cubes and washed in PBS. Lung mononuclear cells were isolated by collagenase enzyme digestion with a solution of collagenase type I (Wako, Osaka, Japan) and bovine pancreatic DNase I (Sigma) in Dulbecco's PBS with Mg2+ and Ca2+ (Sigma). Every 15 min the tissue suspension was aspirated gently and expelled from a syringe through a 19-gauge needle to assist tissue disruption, agitated thoroughly with shaking for 1 h at 37°C and finally passed through a nylon mesh strainer (Falcon, Franklin Lakes, NJ, USA) to exclude large fragments. The isolated cell suspension was centrifuged at 350 g for 10 min at 5°C, the cell pellet was resuspended in ammonium chloride lysing buffer (Biosource International, Camarillo, CA, USA) and the samples were then processed for cell counting. The cells were pre-incubated with anti-Fcγ RIII antibody (BD PharMingen) on ice for 15 min in PBS containing 1% fetal calf serum and then stained with FITC-conjugated anti-CD3ε antibody and phycoerythrin (PE)-conjugated anti-pan NK antibody (DX-5) (BD PharMingen), PE-conjugated anti-IFN-γ antibody and FITC-conjugated anti-very late activating antigen-4 (VLA-4) antibody (eBiosciences, San Diego, CA, USA). Isotype-matched irrelevant antibodies were used as the control antibody. Approximately 10 000 cells were analysed by a fluoresence activated cell sorter (FACScan; BD Biosciences, Mountain View, CA, USA) and fluorescence intensity was expressed on a log scale from 1 to 103. For detection of intracellular IFN-γ, the cells were permeabilized by a permeabilization buffer (eBioscience, SanDiego, CA, USA), according to the manufacturer's procedure.

Extraction of RNA and reverse transcription–polymerase chain reaction

Lungs were snap-frozen in liquid nitrogen and stored at −80°C for RNA analyses. Total cellular RNA was extracted from the lungs after various time-periods using Trizol reagent (Life Technologies, Grand Island, NY, USA). Phase separation was achieved by adding 200 μl chloroform to the suspension and centrifuging at 12 000 g. After mixing with isopropanol, centrifuging and washing with ethanol, the RNA pellet was resuspended in 30 ml of nuclease free water and further DNAase (20 U/ml) was added. The mixture was incubated for 30 min at 37°C. RNA extracted from lung tissues was reverse-transcribed into cDNA using the RT system with random hexamers (Toyobo, Tokyo, Japan).

Real-time polymerase chain reaction

The expression of ICAM-1, VCAM-1, VLA-4, E-selectin, TNF-α and interleukin (IL)-6 were analysed quantitatively with the real-time polymerase chain reaction (PCR) technique.

The primer sequences were as follows: for VCAM-1 mRNA: sense, 5′-CAATGGGGTGGTAAGGAA-3′, anti-sense, 5′- GTCACAGCGCACAGGTAAGA-3′; for ICAM-1 mRNA, sense, 5′-GAG AGTGGACCCAACTGGAA-3′, anti-sense, 5′- GCCACAGTTCTCAAAGCACA-3′; for E-selectin mRNA, sense, 5′-AGTCTAGCGCCTGGATGAAA-3′, anti-sense, 5′- CCAGCGAGGAGAACAAAAAC-3′; for VLA-4 mRNA, sense, 5′-AATGCCTCAGTGGTCAATCC-3′, anti-sense, 5′-CTACCCAGCTGGAGCTGTTC-3′; for IL-6 mRNA, sense, 5′- GTTCTCTGGGAAATGGTGGA-3′, anti-sense, 5′-TTCTGCAAGTGCATCATCGT-3′; and for TNF-α mRNA, sense, 5′-TGTTGCCTCCTCTTTTGCTT-3′, anti-sense, 5′-TGGTCACCAAATCAGCGTTA-3′. Real-time PCR was run in sealed 96-well microplates. The reaction mixture consisted of cDNA, primers and SYBER Green Master Mix (Toyobo) for a final volume of 20 μl. Following 2-min incubation at 50°C and 5-min incubation at 95°C, the reaction ran for 40 cycles. Each cycle consisted of a 15-s denaturing phase at 95°C and a 1-min annealing-extension phase at 60°C. The resulting change in fluorescence was measured using Applied Biosystems 7700 Prism Sequence Detector (PE Applied Biosystems, Foster City, CA, USA). Data were analysed with Sequence Detector version 1·6 included with the 7700 Detector. The relative amount of transcript was determined using the comparative Ct method according to the manufacturer's instructions. The cytokine mRNA levels in each sample are expressed as the fold increase over non-treated controls in the ratio of each target gene to actin. The gene expression level of iNOS and IFN-γ in the lung was examined by standard PCR. The primers used for detection of iNOS mRNA were: sense, 5′-TCTTGACCATCAGCTTGCAA-3′, anti-sense, 5′-TCTTTGACGCTCGGAACTGT-3′; and for IFNγ mRNA: sense, 5′-CTCAAGTGGCATAGATGT-3′, anti-sense, 5′-GAGATAATGTGGCTCTGCAGGATT-3′. The reaction was performed on a DNA thermal cycler (Perkin-Elmer, Cetus, CA, USA) for 25 cycles; denaturation was carried out at 94°C for 30 s min, annealing at 55°C for 45 s and extension at 72°C for 1 min. Glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified as a control. Care was taken to prevent cross-contamination and carry-over of PCR products. PCR products were run on a 1·5% agarose gel and stained with ethidium bromide.

Determination of cytokine level in bronchoalveolar lavage fluid

Bronchoalveolar lavage fluid (BALF) was obtained by washing with 600 μl of Hanks' balanced salt solution in portions of 200 μl via a tracheal incision. After centrifuging, the cell pellets were resuspended in PBS and stained with Giemsa's solution. The cells were identified microscopically as mononuclear cells (primarily alveolar macrophages), neutrophils or other cells. Supernatants were preserved at −80°C for the measurement of total protein concentration and levels of TNF-α, IL-1β and IL-6. The level of each cytokine was measured with enzyme-linked immunosorbent assay (ELISA) kits (TNF-α and IL-1β kits from R&D company; IL-6 from Biosciences).

Quantification of pulmonary microvascular protein leakage

The solution containing 0·4% Evans blue dye (50 mg/kg) was injected i.v. Thirty minutes later, the mice were killed and the pulmonary circulation was flushed with PBS. The lungs were removed and snap-frozen in liquid nitrogen. The frozen tissue was homogenized in PBS, and Evans blue dye was extracted into formamide by incubating at 60°C for 16 h. Absorbance of the supernatant was measured at 620 and 740 nm. Extravasated Evans blue dye concentration (μg Evans blue per lung in min) in lung homogenate was calculated against a standard curve as described elsewhere [12].

Statistical analysis

Experimental values are expressed as the mean ± standard deviation in at least three independent experiments. Significant differences between experimental and control groups were determined with Student's t-test. A value of P < 0·01 was considered statistically significant.

Results

Sensitization process for lethal endotoxic shock

Sensitization with α-GalCer induces the increase of activated NK T cells and production of IFN-γ in the lung

First, we examined the cell types of infiltrates increasing in the lungs of α-GalCer-sensitized mice. α-GalCer (1 μg) was injected i.v. into BALB/c mice and 12 h later the lungs were removed. As shown in Fig. 1a, laser flow cytometric analysis of pulmonary intraparenchymal leucocytes demonstrated that NK T cells expressing pan-NK marker and CD3 increased in the lungs of α-GalCer-sensitized mice. Conversely, there was only a small number of such cells in the lungs of normal control mice. The increase of NK T cells expressing both NK cell and T cell markers was reasonable, because α-GalCer is the ligand stimulating NK T cells [9,10]. FACS analysis also demonstrated that NK T cells present in the lungs of α-GalCer-sensitized mice were larger cells with higher forward-scatter than cells from normal control mice (data not shown), suggesting the presence of activated NK T cells in the lungs of α-GaCer-sensitized mice.

Fig. 1.

Fig. 1

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Production of interferon (IFN)-γ production by natural killer (NK) T cells in lungs of α-galactosylceramide (α-GalCer)-sensitized mice. (a) Laser flow cytometric analysis of NK T cells from lungs of α-GalCer-sensitized mice. The cells from lungs of mice injected with α-GalCer (1 μg) 12 h before were stained with phycoerythrin (PE)-conjugated anti-pan NK antibody and fluorescein isothiocyanate-conjugated anti-CD3 antibody. The fluorescence intensity is expressed in a log scale. (b) The IFN-γ mRNA in lungs of mice injected with α-GalCer 3, 6, 9 and 12 h before was analysed by reverse transcription–polymerase chain reaction. (c) IFN-γ-producing cells were analysed by laser flow cytometry. The cells from lungs of mice injected with α-GalCer 12 h before were permeabilized and stained with PE-conjugated anti-IFN-γ antibody. The fluorescence intensity is expressed in a log scale. A typical experiment of three experiments is shown.

α-galactosylceramide is well known to stimulate NK T cells and induce IFN-γ production [13,14], and a significant level of IFN-γ is reported to circulate in α-GalCer-sensitized mice [8,15–17]. In the preceding section, α-GalCer pre-treatment led to an increase of NK T cells in the lung. Therefore, we examined whether IFN-γ was produced locally in the lungs of α-GalCer-sensitized mice (Fig. 1b). Mice were injected i.v. with α-GalCer and the lungs were removed 3, 6, 9 and 12 h after the treatment. The levels of IFN-γ mRNA and protein were determined by reverse transcription–polymerase chain reaction (RT–PCR) and FACS analysis respectively. RT–PCR analysis demonstrated that IFN-γ mRNA was detected in the lungs 3 h after the injection of α-GalCer and the IFN-γ mRNA expression continued up to 12 h. No IFN-γ mRNA was detected in mice injected with either saline or LPS alone.

Further, we examined the production of IFN-γ in pulmonary intraparenchymal leucocytes. The permeabilized cells were stained with PE-conjugated anti-IFN-γ antibody and analysed by laser flow cytometry (Fig. 1c). FACS analysis demonstrated a significant increase of IFN-γ-producing cells in the lungs of α-GalCer-pretreated mice. Thus, α-GalCer was suggested to induce the local IFN-γ production via stimulation of NK T cells increasing in the lungs of α-GalCer-sensitized mice.

Local IFN-γ production induces expression of VCAM-1 in the lung of α-GalCer-sensitized mice

During the experiments, we found the accumulation of a significant number of inflammatory cells in the lungs of α-GalCer-sensitized mice. The cell numbers in BALF from α-GalCer-treated and non-treated mice were approximately 51 000 ± 1000/lung and 21 000 ± 500/lung respectively, and approximately 85% of the infiltrates were mononuclear cells. Therefore, we examined the participation of adhesion molecules in recruitment of the infiltrates and, further, the relationship between the production of IFN-γ and the expression of adhesion molecules in the lung. The lungs were removed at various times from α-GalCer-sensitized mice and RNA was extracted from the lungs. The mRNA levels of ICAM-1 (CD54), E-selectin (CD62E) and VCAM-1 (CD106) were analysed by real-time PCR. As shown in Fig. 2a, VCAM-1 mRNA expression was up-regulated strikingly in the lungs of α-GalCer-sensitized mice. The expression of E-selectin mRNA was augmented. Conversely, ICAM-1 mRNA did not increase in α-GalCer sensitization. Because VCAM-1 mRNA was up-regulated markedly by α-GalCer sensitization, the effect of anti-IFN-γ neutralizing antibody on the augmentation of VCAM-1 expression was examined in the lungs of α-GalCer-sensitized mice (Fig. 2b). The up-regulation of VCAM-1 mRNA expression was inhibited definitively by anti-IFN-γ neutralizing antibody, but not by an isotype-matched control antibody.

Fig. 2.

Fig. 2

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Expression of adhesion molecules in lungs of α-galactosylceramide (α-GalCer)-sensitized mice. (a) The mRNA level of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin from lungs of mice injected with α-GalCer 3–12 h before was analysed by real-time polymerase chain reaction (PCR). *P < 0·01. (b) The effect of anti-interferon (IFN)-γ antibody on the expression of VCAM-1 mRNA in lungs of mice injected with α-GalCer was examined. Anti-IFN-γ antibody and α-GalCer were injected intravenously into mice and 12 h later the VCAM-1 mRNA was extracted. Three mice were used for each experimental group and the experimental data are expressed as the mean ± standard deviation from three independent experiments. *P < 0·01. (c) The localization of VCAM-1 in lungs from mice injected with α-GalCer 12 h previously was detected by immunohistochemical staining with anti-VCAM-1 antibody. Magnification ×400.

Next, the localization of the expression of adhesion molecules was examined immunohistochemically in the lungs of α-GalCer-sensitized mice. Strong expression of VCAM-1 was detected on blood vessels in the lungs 6 h after α-GalCer sensitization and VCAM-1 expression on blood vessels continued at 12 h (Fig. 2c). Expression of ICAM-1 and E-selectin was detected as a faint positive staining. In order to clarify the role of VCAM-1 on the development of LPS-mediated lethal shock, anti-VCAM-1 antibody (250 μg/mouse) was administered i.v. together with α-GalCer into mice. Anti-VCAM-1 antibody significantly prevented the mice from LPS-induced acute lung injury and lethality (three of six mice).

Vascular cell adhesion molecule-1 expression recruits inflammatory cells expressing VLA-4 as the counterpart of VCAM-1 in the lung

Expression of VCAM-1 is known to recruit the cells expressing VLA-4 as the counterpart in the lung [18,19]. Therefore, we examined whether VCAM-1 expression recruited VLA-4-expressing cells into the lungs of α-GalCer-sensitized mice by analysis of VLA-4 mRNA and protein. The RNA fraction was extracted from the lungs at the indicated time after α-GalCer sensitization and was quantified by real-time PCR. As shown in Fig. 3a, the level of VLA-4 mRNA expression was augmented markedly in the lungs after 9 h, and increased further 12 h after α-GalCer sensitization. LPS alone did not affect VLA-4 mRNA expression.

Fig. 3.

Fig. 3

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Detection of very late activating antigen-4 (VLA-4)-positive cells in lungs of α-galactosylceramide (α-GalCer)-sensitized mice. (a) The VLA-4 mRNA level in lungs of mice injected with α-GalCer 3, 6, 9 and 12 h before analysed by real-time polymerase chain reaction. Three mice were used for each experimental group and the experimental data are expressed as the mean ± standard deviation from three independent experiments. *P < 0·01 versus 0 h. (b) The accumulation of VLA-4-positive cells in the lungs of mice injected with α-GalCer 12 h before were analysed by laser flow cytometry with fluorescein isothiocyanate-conjugated anti-VLA-4 antibody. The fluorescence intensity is expressed in a log scale. A typical experiment of three experiments is shown.

Next, pulmonary intraparenchymal leucocytes isolated from the lungs of α-GalCer-sensitized mice were stained with FITC-conjugated anti-VLA-4 antibody in order to quantify the expression of VLA-4 protein. VLA-4 expression was analysed by laser flow cytometry (Fig. 3b). VLA-4 was expressed definitively on infiltrates present in the lungs from α-GalCer-sensitized mice.

Execution process for LPS-mediated lethal shock

Lipopolysaccharide induces the production of proinflammatory cytokines in lungs of α-GalCer-sensitized mice

In the preceding sections we demonstrated that α-GalCer increased NK T cells and induced local production of IFN-γ in the lung, and that the IFN-γ produced led to the expression of VCAM-1 and recruitment of a number of VLA-4-expression cells. A large number of inflammatory cells might respond to LPS and produce proinflammatory mediators in the lungs of α-GalCer-sensitized mice. We examined LPS-induced cytokine production in the lungs of α-GalCer-sensitized mice. LPS (5 μg) was injected i.v. into α-GalCer-sensitized mice and 6 h later the levels of TNF-α, IL-1β and IL-6 in sera, lung tissue and BALF were determined with ELISA. Injection of LPS led to the production of higher levels of TNF-α in lung tissues and BALF from α-GalCer-sensitized mice than in untreated mice (Fig. 4a). A higher level of IL-1β was also detected in sera, lung tissues and BALF in α-GalCer-sensitized mice (Fig. 4b). LPS induced a higher output of IL-6 as well as TNF-α and IL-1β in α-GalCer-sensitized mice (Fig. 4c). In addition, TNF-α and IL-6 mRNA were also up-regulated in the lung tissues after administration of LPS into α-GalCer-sensitized mice (data not shown).

Fig. 4.

Fig. 4

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Production of tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in lungs of mice receiving α-galactosylceramide (α-GalCer) and lipopolysaccharide (LPS). LPS was injected into the mice sensitized with α-GalCer 12 h before. The concentration of cytokines in lung tissues, bronchoalveolar lavage fluid and sera was determined 6 h after LPS injection by enzyme-linked immunosorbent assay. The experimental data are shown as the mean of triplicates ± standard deviation from three independent experiments. *P < 0·01 versus none or LPS.

Lipopolysaccharide induces expression of iNOS and production of nitric oxide in the lungs from α-GalCer-sensitized mice

Several reports support the participation of nitric oxide (NO) and its rapid reaction with superoxide leading to the generation of extremely harmful oxidants, such as peroxynitrite and hydroxyl radicals, in sepsis-induced acute lung injury [20,21]. Therefore, we examined the expression of iNOS mRNA and protein in the lungs of α-GalCer-sensitized mice by the injection of LPS. First, iNOS mRNA expression was analysed by RT–PCR. Administration of LPS induced a marked expression of iNOS mRNA in α-GalCer-sensitized mice, but not in non-treated mice (Fig. 5a), whereas injection of α-GalCer alone did not induce it in untreated mice. Next, the expression of LPS-induced iNOS protein in the lungs was detected by immunohistochemical staining with anti-iNOS antibody (Fig. 5b). Positive staining was detected clearly in the lungs of α-GalCer-sensitized mice. In addition, the NO-mediated tissue damage was examined by immunohistochemical staining with anti-nitrotyrosine antibody. Positive staining was detected in the lungs of α-GalCer-sensitized mice (data not shown).

Fig. 5.

Fig. 5

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Expression of inducible nitric oxide synthetase (iNOS) in lungs of mice receiving α-galactosylceramide (α-GalCer) and lipopolysaccharide (LPS). LPS was injected into the mice sensitized with α-GalCer 12 h before. (a) The level of iNOS mRNA in the lungs 6 h after LPS injection was analysed by reverse transcription– polymerase chain reaction. (b) The localization of iNOS in lungs of the mice was determined by immunohistochemical staining with anti-iNOS antibody. A typical experiment of three experiments is shown. Magnification ×400.

Lipopolysaccharide induces enhanced pulmonary permeability in the lungs of α-GalCer-sensitized mice

We tried to quantify LPS-induced acute lung injury by leakage of proteins and Evans blue dye into BALF. The protein level in BALF was determined by administration of LPS into α-GalCer-sensitized mice (Fig. 6a). Administration of LPS caused the leakage of a large amount of proteins into BALF in α-GalCer-sensitized mice, but not in untreated mice. Cell numbers in BALF increased significantly compared with those in non-treated mice.

Fig. 6.

Fig. 6

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Elevation of pulmonary leakage in lungs of mice receiving α-galactosylceramide (α-GalCer) and lipopolysaccharide (LPS). LPS was injected into the mice sensitized with α-GalCer 12 h before. (a) The protein concentration in bronchoalveolar lavage fluid (BALF) was determined 6 h after LPS injection by a colorimetric assay. Bovine serum albumin was used as a standard protein. (b) The number of cells in BALF was counted 6 h after LPS injection in the mice. (c) Pulmonary permeability was determined 6 h after LPS injection by leakage of Evans blue dye. The content of Evans blue in the lung is expressed as Evans blue (μg)/lung (g)/min. All figures are shown as the mean of triplicates ± standard deviation from three independent experiments. *P < 0·01 versus none or LPS.

Next, we examined the effect of LPS on pulmonary permeability in the lungs of α-GalCer-sensitized mice (Fig. 6b). The leakage of Evans blue dye into BALF was determined quantitatively with a spectrophotometer. LPS led to the leakage of a higher concentration of Evans blue dye detected in BALF from α-GalCer-sensitized mice than from untreated mice (Fig. 6c).

Discussion

In the present work, we studied the mechanism of development of acute lung injury in LPS-mediated lethal shock using α-GalCer-sensitized mice. α-GalCer leads to the increase of NK T cells and local production of IFN-γ in the lung at an initial stage after the injection. Subsequently, IFN-γ induces the expression of VCAM-1 on blood vessels in the lung, followed by infiltration of VLA-4-positive cells as the counterpart of VCAM-1. The α-GalCer sensitization may be completed by the accumulation of inflammatory cells, mainly VLA-4-positive cellls, into the lung. The presence of a large number of infiltrates in the lung might be prepared for LPS challenge. Administration of LPS into α-GalCer-sensitized mice causes the production of excessive proinflammatory cytokines and mediators, such as TNF-α, IL-1β, IL-6 and NO, in the lung and various cell types undergo apoptosis there. Acute lung injury because of massive cell death and elevated pulmonary permeability results finally in respiratory failure. This is the possible mechanism of development of respiratory failure in LPS-mediated lethal shock.

Natural killer T cells increased in the lung 3–12 h after α-GalCer treatment; originally, only a small number of NK T cells were present [22,23]. However, the significant increase of NK T cells could not be explained only by proliferation of NK T cells present in the lung, because of the lack of sufficient doubling time. Therefore, stimulation of NK T cells originally present in the lung with α-GalCer may cause the production of some chemotactic factor for NK T cells [24,25], and a significant number of NK T cells may be recruited into the lung. Alveolar macrophages are unlikely to participate in chemotactic factor production because α-GalCer does not stimulate macrophages directly [26]. The precise mechanism of how α-GalCer increases NK T cells in the lung is still unclear.

Natural killer T cells produce IFN-γ in response to α-GalCer [27,28]. Recently, we have demonstrated that IFN-γ plays a critical role in the development of LPS-mediated lethal shock because anti-IFN-γ neutralizing antibody prevents lethal shock [8]. However, the precise action of IFN-γ was unknown in the previous report. The present study suggests that IFN-γ induces the expression of adhesion molecules, especially VCAM-1, in the lung. Anti-IFN-γ neutralizing antibody prevented the increase of VCAM-1 mRNA. Moreover, anti-VCAM-1 antibody prevented LPS-mediated lethal shock in α-GalCer-sensitized mice (50% inhibition). Therefore, IFN-γ-induced VCAM-1 expression might be a pivotal event in α-GalCer sensitization. NK T cells also produce IL-6, IL-4 and IL-1β, as well as IFN-γ, in response to α-GalCer. The possibility is not excluded that those cytokines might play a role in the expression of adhesion molecules and accumulation of inflammatory cells in the lung.

Previously we reported the pivotal role of TNF-α in LPS-mediated lethal shock [29,30]. The present study demonstrates that TNF-α is produced locally in the lung. LPS possibly causes the local release of TNF-α via stimulation of infiltrates in the lung. An excess of TNF-α may lead to damage of vascular endothelial cells in the lung, followed by elevated pulmonary permeability, bleeding and finally respiratory failure. In addition, IL-1β, IL-6 and NO are also produced in the lung of α-GalCer-sensitized mice. Although reported to be involved in the development of acute lung injury in other experimental models [31,32], the respective roles of IL-1β, IL-6 and NO in our model are not yet clear.

Acute lung injury in septic shock is characterized by elevated permeability of the pulmonary microvascular endothelial cell barrier, resulting in the leakage of protein-rich plasma fluid and circulating inflammatory cells into the pulmonary interstitium and air spaces [33,34], consistent with the development of acute lung injury in our experimental model with α-GalCer sensitization. The pathophysiological changes occurring in the lung of α-GalCer-sensitized mice displays key features of acute lung injury, including inflammation, pulmonary oedema and mortality in septic shock. Therefore, our experimental model provides a useful tool to characterize the pathogenesis and treatment of clinical respiratory failure in patients with sepsis and septic shock.

Neither simultaneous administration of LPS with α-GalCer nor administration of LPS 48 h after α-GalCer sensitization was lethal for mice [8]. Further, mice treated with α-GalCer survived when injected within 2 h before and after LPS challenge [35]. This survival has been explained by low levels of T helper 1 (Th1) cytokines, such as IFN-γ and TNF-α, and by high levels of Th2 cytokines, such as IL-4 and IL-10 [35]. However, the present study demonstrates no sufficient accumulation of NK T and inflammatory cells in the lung for cytokine production 2 h after α-GalCer treatment. The lung is not yet prepared for LPS challenge, which causes acute lung injury. Moreover, the administration of α-GalCer 2 h after LPS challenge is unlikely to lead to acute lung lesions because of no preparation for LPS challenge. Taken together, the present study indicates that α-GalCer sensitization requires a given time to cause acute lung lesions and that an insufficient time-period between α-GalCer and LPS challenge does not cause lethal respiratory failure in mice.

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 and the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan. We are grateful to K. Takahashi for the technical assistance. We thank the Kirin Brewery Company for providing synthetic α-GalCer.

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