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Published ahead of print on December 23, 2003, doi:10.1165/rcmb.2003-0197OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 808-815, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0197OC

CD40 Plays a Crucial Role in Lipopolysaccharide-Induced Acute Lung Injury

Naozumi Hashimoto*, Tsutomu Kawabe*, Kazuyoshi Imaizumi, Toru Hara, Masakazu Okamoto, Katsuyuki Kojima, Kaoru Shimokata and Yoshinori Hasegawa

Department of Medicine, Division of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan

Address correspondence to: Tsutomu Kawabe, M.D., Ph.D., Department of Medicine, Division of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466–8550, Japan. E-mail: kawabet{at}med.nagoya-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activated alveolar macrophages (AM{phi}) are known to constitute a critical modulator of the lung inflammatory response through the production of various mediators. However, the role of activated AM{phi} in acute lung injury (ALI) and acute respiratory distress syndrome is less well known. To address this issue, we examined a lipopolysaccharide (LPS)-induced lung injury model for the role of activated AM{phi} in vivo, focusing on activation through CD40, which is one of the most important pathways for the activation of antigen-presenting cells. Without CD40, LPS-induced ALI was significantly reduced in its histological degree of injury and recruitment of neutrophils into the lung. In addition, the release in the lung of inflammatory mediators such as tumor necrosis factor-{alpha}, interleukin-1ß, macrophage inflammatory protein 2, or matrix metalloproteinase was significantly reduced in mice deficient in CD40 (CD40KO). To elucidate the mechanism of this attenuation of ALI in CD40KO mice, we studied the function of AM{phi} ex vivo. AM{phi} purified from CD40KO mice could not induce expression of inducible nitric oxide synthase (iNOS) by LPS, although iNOS in wild-type AM{phi} was induced by LPS independently of CD40–CD154 interaction. The loss of surface expression of CD40 was enough to interrupt the expression of iNOS in AM{phi} in response to LPS. Also based on the tissue nitrotyrosine staining, the reactive oxygen and nitrogen intermediates seemed to be reduced in tissue in CD40KO mice. These results indicated that activation of AM{phi} through CD40 might be involved not only in amplification by the interaction with CD154 but also in the development of ALI by CD40 itself, and that the functional blockade of CD40 would yield one of the targets for the treatment of LPS-induced ALI and acute respiratory distress syndrome.

Abbreviations: 3-amino-9-ethylcarbazole, AEC • acute lung injury, ALI • alveolar macrophages, AM{phi} • antigen-presenting cells, APC • acute respiratory distress syndrome, ARDS • bronchoalveolar lavage, BAL • deficient in CD40, CD40KO • enzyme-linked immunosorbent assay, ELISA • interferon, IFN • interleukin, IL • lipopolysaccharide, LPS • macrophage inflammatory protein 2, MIP-2 • matrix metalloproteinase, MMP • nitric oxide, NO • inducible NO synthase, iNOS • phosphate-buffered saline, PBS • polyvinylidene difluoride, PVDF • scavenger receptor A, SRA • TNF receptor-associated factor, TRAF • toll-like receptor, TLR • tumor necrosis factor-{alpha}, TNF-{alpha} • tyramide signal amplification, TSA • wild-type, WT


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are still major causes of mortality in intensive care units even though it was reported that a clinical trial of a ventilation strategy involving low tidal volumes reduced mortality by 22 percent (1). Pathologic changes in ARDS include diffuse alveolar damage with neutrophils, macrophages, erythrocytes, hyaline membranes, and disruption of pulmonary capillary integrity leading to protein-rich edema fluid in the alveolar spaces. The majority of these pathologic features of human ARDS have also been observed in experimental lung injury in animals.

Endotoxin or lipopolysaccharide (LPS) is a glycolipid that constitutes the major portion of the outermost membrane of gram-negative bacteria, and is capable of inducing severe lung injury in gram-negative bacterial sepsis and pneumonia, which are among the most common predisposing causes of ARDS (2). The interaction of the lipid A moiety of LPS with macrophages, especially alveolar macrophages (AM{phi}) in the lung, appears to be especially important because subsequent cellular activation results in the release of inflammatory mediators and phenotypic changes. As well as providing the first line of defense against inhaled organisms and irritants, AM{phi} in the lungs are known to be a critical modulator of that organ's inflammatory response through the production of various proinflammatory and anti-inflammatory cytokines in addition to their phagocytic clearance role in activation (3, 4). As inflammatory mediators, AM{phi} could release (i) systemically active proinflammatory cytokines and chemokines, including tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1ß, and macrophage inflammatory protein (MIP)-2 (4); and (ii) reactive oxygen and nitrogen intermediates, including superoxide anion, hydrogen peroxide, hydroxyl radicals, and nitric oxide (NO), which in turn mediate systemic toxicity (4). What role the activation of AM{phi} plays in the development and amplification of lung injury remains unclear.

Activation of AM{phi} also enhances the expression of several molecules, including major histocompatibility complex (MHC), adhesion molecules, and costimulatory molecules (4). CD40 is one of the receptors on AM{phi}, and the expression is upregulated after activation by soluble factors as reported previously in our laboratory (5). Activation induced by CD40–CD154 (CD40 ligand) interaction is one of most important pathways for the activation of antigen-presenting cells (APC).

CD40 is a cell surface receptor that belongs to the TNF-receptor family and was first identified and functionally characterized on B lymphocytes. Recent reports indicate that the CD40/CD40L is expressed in nonhematopoietic cells, including endothelial and epithelial cells (6). As numerous recent studies have demonstrated, the CD40 ligation on APC is associated with the augmentation of inflammation as follows (6): (i) enhanced survival of these cells; (ii) secretion of cytokines (such as IL-1, IL-6, IL-8, IL-10, IL-12, TNF-{alpha}, MIP-1{alpha}) and enzymes such as matrix metalloproteinases (MMPs); (iii) enhanced monocyte tumoricidal activity; and (iv) NO synthesis. The CD40–CD154 interaction in the lung was the critical step for regulating inflammation and fibrosis in experimental models induced by antigen, oxygen, radiation, or soluble CD154 (7–10), and blocking its interaction prevents pulmonary inflammation. Although these findings suggested that CD40 ligand–dependent AM{phi} activation or the production of inflammatory mediators might be the other important mechanism in the induction of ALI, the role of CD40 on activated AM{phi} in response to LPS has remained unclear.

In this study, we examined an LPS-induced lung injury model in the role of activated AM{phi} in vivo and focused on activation induced by a CD40–CD154 interaction using mice deficient in CD40 (CD40KO). Such mice showed attenuation of the degree of ALI at any point in the histologic estimation, cellular characterization, and analysis of the inflammatory mediators in the lung. To elucidate the role of CD40 on activated alveolar macrophages for the development and amplification of lung injury, we also evaluated the function of AM{phi} ex vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
LPS (Escherichia coli 055:B5) were obtained from BD Diagnostic Systems (Sparks, MD). Histochoice MB was from Amresco (Solon, OH). The Tyramide signal amplification (TSA) biotin system was from Perkin Elmer Life Science, Inc. (Boston, MA). Mouse interferon (IFN)-{gamma} (mIFN-{gamma}) were from Invitrogen Corp. (Carlsbad, CA). Enzyme-linked immunosorbent assay (ELISA) Development Kits for mouse IL-1ß, mouse TNF-{alpha}, mIFN-{gamma}, and mouse IL-12 were obtained from Genzyme Techne Corporation (Minneapolis, MN). Quantikine M Mouse MIP-2 Immunoassay was from R&D Systems, Inc. (Minneapolis, MN). Rat anti-mouse CD40 IgG (3/23) and rat anti-mouse CD14 IgG (rmC5–3) were from BD PharMingen (San Diego, CA). Polyclonal rabbit anti–inducible NO synthase (iNOS) Ab was from BD Transduction (Lexington, KY). Rat anti-mouse macrophage scavenger receptor A (SRA) IgG (2F8) was from Serotec Ltd (Oxford, UK). Polyclonal rabbit anti-nitrotyrosine Ab was from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit biotinylated anti-rat IgG (H+L) and 3-amino-9-ethylcarbazole (AEC) immunostaining kit was obtained from Vector Laboratories (Burlingame, CA). Peroxidase-linked anti-rabbit IgG and Rainbow marker were from Amersham Pharmacia Biotech, Inc. (Buckinghamshire, UK). Peroxidase-linked anti-rat IgG was from DakoCytomation (Glostrup, Denmark). Coomassie Brilliant Blue R 250 (CBBR250) was from Fluka (Buchs, Switzerland).

Animals and Experimental Model of ALI
BALB/c CD40KO mice backcrossed for more than seven generations with BALB/c mice were kindly provided by Dr. Hitoshi Kikutani (Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Japan). BALB/c mice (wild-type [WT] mice) were purchased from SLC (Shizuoka, Japan) and used as WT mice. Six- to 12-wk-old WT mice and CD40KO mice matched for age, sex, and weight were used for the studies. After being anesthetized with pentobarbital sodium, each WT and CD40KO mouse was injected intratracheally with either 0.2 mg/kg LPS in phosphate-buffered saline (PBS) or endotoxin-free PBS alone (11).

Collection and Measurement of Specimens
After 24 h, mice were exsanguinated with ether anesthetization followed by aortic perforation. Bronchoalveolar lavage (BAL) fluid, blood, and lung tissue were then collected. Blood samples were obtained by retroorbital bleeding, and blood smears were prepared by staining with May-Gruenwald and Giemsa solution (Merck, Darmstadt, Germany). Serum was obtained by centrifugation after keeping samples for 1 h at 4°C. To collect BAL fluid, the trachea was cannulated, the lungs were lavaged six times with PBS (0.5 ml each time), and ~ 2.5 ml of the instilled fluid was consistently recovered. Total cell numbers were counted with a standard hemocytometer. After centrifugation, supernatants were stored at –80°C for cytokine measurements by ELISA and MMP activities assessed by zymography, and cell pellets were used to prepare cytospins. Smears of BAL cells were prepared with cytocentrifugation using a Cytospin 2 (Shandon Inc., Cheshire, UK) at 1,000 rpm for 5 min and then stained with May-Gruenwald and Giemsa solution. Cell differentiation was examined by counting at least 200 cells using standard hemocytologic criteria to classify the cells as neutrophils, eosinophils, lymphocytes, or monocytes/macrophages. For regular staining with hematoxylin and eosin for morphologic analysis and immunostaining, the lungs of mice in each group not subjected to BAL administration were removed from the thoracic cavity and cleared of extraneous tissue. The wet lung weight was measured as an indicator of lung inflammation (12). The wet lung-to-body weight ratio was then calculated. Serial slices from apex to base of the lung samples of each mice group were prepared and fixed in 4% paraformaldehyde on ice for 6 h, dehydrated through graded sucrose washes for 24 h, and finally fixed in OCT compound (Tissue-Tek; Miles, Elkhart, IN). To assess the degree of lung damage, the lungs were obtained in WT and CD40KO mice (four animals in each category). Sections were submitted to three examiners who were blinded as to animal groups. Five fields chosen randomly from each section (a total of 20 fields per animal) were examined at a magnification x100. Quantal assessment of injury was performed in blinded fashion by grading three histologic findings: hemorrhage, inflammation, and edema. A separate grade from 1 (normal) to 5 (diffuse abnormality) was calculated for each field depending on the following criteria: (i) the extravasation of fluid, red blood cells, or inflammatory cells; and (ii) the presence of polymorpho- or mononuclear cells. For each variable, a single score was calculated per animal by summing the field scores. The total lung injury score was calculated as the sum of the three components. Whole lungs were also homogenized after killing for Western blotting with six volumes of ice-cold tissue lysis buffer consisting of 50 mM Tris base-pH 6.8, 2% sodium dodecyl sulfate (SDS), 5 mM ethylenediaminetetraacetic acid (EDTA) and Complete, EDTA-free (Roche Applied Science, Penzberg, Germany). Homogenized samples were centrifuged for 40 min at 10,000 x g at 4°C twice, and aliquots were analyzed for total protein concentration by the Bradford assay using bovine serum albumin as the standard. Ten percent glycerol, 0.005% bromophenol blue, and 5% ß-mercaptoethanol were added to the resulting supernatants and then frozen until use. The intensities were estimated by the public domain NIH image 1.61 program (13).

Cytokine and Chemokine Assay
TNF-{alpha}, IL-1ß, IFN-{gamma}, IL-12, and MIP-2 concentrations in BAL fluid, serum, and culture supernatants were determined by ELISA. All procedures were performed according to the manufacturer's instructions.

Gelatin Zymography
To characterize the activity of MMPs in BAL fluids, samples were analyzed by gelatin zymography as described previously (14). Briefly, SDS-PAGE was performed using 10% polyacrylamide gels. The gels for examining gelatinase activity were prepared by including gelatin in the running gel (1 mg/ml final concentration). The samples were diluted with nonreducing sample buffer and not boiled before electrophoresis. The gels were cooled to 4°C during electrophoresis, after which they were washed twice in 10 mM Tris-HCl, 2.5% Triton X-100 (vol/vol), and pH 8.0 for 30 min. After the gels were rinsed for 15 min in the above buffer without Triton X-100, they were incubated for 18 h at 37°C in a buffer containing 50 mM Tris-HCl, 10 mM CaCl2, and 0.02% NaN3, pH 7.5. Following the incubation, the gels were stained with 0.1% CBBR250 in 50% methanol/10% acetic acid (vol/vol) and destained in 10% methanol/10% acetic acid (vol/vol). Negative staining indicated zones of enzymatic activity. The molecular weights of the gelatinolytic bands were estimated by Rainbow marker. The intensities of MMP-9 bands were estimated by the public domain NIH image 1.61 program (13).

AM{phi} Preparation
Naive mice were exsanguinated with ether anesthetization followed by aortic perforation. AM{phi} were isolated by BAL as described above. Lavaged cells from both WT mice and CD40KO mice were respectively pooled followed by cell counts and differential cell analysis. By morphologic estimation with Giemsa staining, it was confirmed that pooled cells from each mouse consisted of > 98% AM{phi} for separate experiments. Cells were resuspended in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 10% fetal calf serum, which we used as a complete medium. After allowing the cells to adhere to the plates for 3 h, nonadherent cells were removed with three washes. We used the adhesive cells as AM{phi}. The cells were resuspended in the complete medium, plated at 5 x 105/ml into a 96-well Lab-Tek Chamber Slide System (Nalge Nunc International, Naperville, IL), and treated according to the following experiments. AM{phi} were incubated with or without 0.1, 1, or 10 µg/ml LPS, or 300 U/ml mIFN-{gamma} for 24 h. In separate experiments, AM{phi} were also incubated with 10 µg/ml LPS for different incubating times of 0, 6, or 24 h. At the conclusion of the respective experiments, the culture supernatants were harvested and stored at –80°C until assay, while the cells were air-dried and fixed in Histochoice MB solution for 20 min for immunocytochemistry as follows. The nitrite concentration in the culture supernatants was measured with a calorimetric assay based on the Griess reaction previously described (15). Briefly, 50 µl of supernatant was incubated for 10 min at room temperature with an equal volume of Griess reagent containing 0.5% sulfanilamide and 0.05% N-(1-naphthyl) ethyleneamine dihydrochloride in 2.5% phosphoric acid. The optical density at 570 nm was measured with a microtiter plate reader. The nitrite contents were quantified by comparison with a standard curve generated with NaNO2 in the range of 0–100 µM.

Immunohistochemistry and Immunocytochemistry
Immunohistochemistry and immunocytochemistry procedures were done using the TSA biotin system as described by the manufacturer with some modification. Incubation and wash procedures were performed at room temperature unless otherwise specified. The washing procedure was done in PBS for 5 min three times unless otherwise specified. Serial cryostat sections (4 µm thick) of the lung specimens were cut onto silanized slides (DAKO Japan Co., Ltd., Kyoto, Japan), air-dried, and fixed in Histochoice MB solution for 20 min. Slides were washed to remove OCT compound. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in methyl alcohol for 30 min, and then washed. The slides for iNOS and nitrotyrosine immunostaining were immersed in 0.2% Triton X-100 in PBS for 15 min, followed by washing. After blocking nonspecific protein binding with blocking buffer (called TNB buffer by the manufacturer) composed of 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.5% Blocking Reagent (supplied in a kit), sections were incubated overnight at 4°C with appropriate dilutions of specific primary antibodies as follows: either rat anti-mouse CD40 IgG, rat anti-mouse CD14 IgG, rat anti-mouse SRA IgG, polyclonal rabbit anti-nitrotyrosine Ab, polyclonal rabbit anti-iNOS Ab, or normal rat or rabbit IgG as controls. The slides were washed and then incubated with appropriate dilutions of rabbit biotinylated anti-rat IgG (H+L) or peroxidase-linked anti-rabbit IgG for 2 h, followed by washing. The slides for CD40, CD14, and SRA immunostaining were incubated with streptavidin–peroxidase complex (HRP-SA) at a dilution of 1:100 in TNB buffer for 30 min, followed by washing. All slides were then incubated for 5 min with biotinyl tyramide diluted at 1: 50 in amplification diluent (supplied in a kit). After washing, the slides were incubated with HRP-SA diluted at 1: 100 in TNB buffer for 30 min. After washing again, an AEC immunostaining kit was used to visualize antibody binding, and then counterstaining with a 1% methyl green stain solution (Muto Pure Chemicals Ltd., Tokyo, Japan) was performed. The sections were mounted with Geltol aqueous mounting medium (Shandon, Pittsburgh, PA).

Western Blotting for Nitrotyrosine, CD14, and SRA in Whole Lung Homogenates
Protein extracts at a concentration of 150 µg/lane were denatured by heating at 95°C for 5 min and separated on 10% SDS-PAGE gels. Electrophoresis was performed at 25 mA followed by transfer of protein to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA). The transferred membranes were blocked with 5% skim milk in TBST solution (0.02 M Tris Base-pH 7.5, 0.5 M sodium chloride; Sigma, and 0.1% of Tween 20; Katayama Chemical, Inc., Osaka, Japan) at room temperature for 1 h and then probed with the same polyclonal rabbit anti-nitrotyrosine Ab (1:1,000), rat anti-mouse CD14 IgG (1:500), and rat anti-mouse SRA IgG (1:500) as previously described, diluted in the blocking solution. Membranes were subsequently rinsed in TBST for 5 min three times and exposed to the secondary HRP-conjugated anti-rabbit IgG for anti-nitrotyrosine Ab or HRP-conjugated anti-rat IgG for CD14 and SRA for 1 h at RT. A chemiluminescence detection system (ECL; Amersham Bioscience) and Hyperfilm (Amersham Bioscience) were used to detect the binding of this antibody.

Statistical Analysis
The results were analyzed by Mann-Whitney test for a comparison between the two groups and by standard one-way ANOVA with post hoc Bonferroni-Dunn test for multiple comparisons. P < 0.05 was considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tolerance to LPS Challenge in CD40KO Mice
When challenged with LPS intratracheally, both WT and CD40KO mice showed serious appearance compared with saline-challenged mice. However, their appearance differed greatly in severity. Whereas LPS-challenged WT mice (LPS WT mice) showed a decrease in spontaneous movement, low reaction to threatening stimuli, short rapid respiration, white periconjunctival exudates, and maintenance of a hunched posture, LPS-challenged CD40KO mice (LPS CD40KO mice) showed these signs only very mildly (Figure 1A). To clarify whether these findings are due to lung inflammation, we estimated the wet lung-to-body weight ratio, which was correlated with the severity of lung injury as previously reported (12). Ratio in LPS WT mice was significantly higher than in saline-challenged WT mice, indicating that LPS administration induced ALI effectively (LPS WT, 0.019 ± 0.002; saline WT, 0.015 ± 0.001; P < 0.05). In contrast, when CD40KO mice were challenged with LPS, the wet lung-to-body weight ratio was significantly lower than that of LPS WT mice (LPS WT, 0.019 ± 0.002; LPS CD40KO, 0.016 ± 0.001; P < 0.05) (Figure 1B). These data suggested that CD40 might be involved in the development and amplification of LPS-induced lung injury.



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  		Figure 1. Phenotypic changes in WT and CD40KO mice with LPS-induced lung injury. (A) Different appearance in WT mice and CD40KO mice due to LPS. WT mice (left) and CD40KO mice (right) were injected intratracheally with LPS (0.2 mg/kg). After 24 h, LPS-injected WT mice showed severely decreased spontaneous movement, decreased reaction to threatening stimuli, tachypnea, white periconjunctival exudates, and maintenance of a hunched posture, whereas CD40KO mice showed only mild signs. Representative figures are shown from three separate experiments. (B) Changes in wet lung-to-body weight ratio after 24 h of LPS or saline injection in WT and CD40KO mice. Data represent the mean ± SEM for groups of five mice. *Significant difference (P < 0.05) between LPS WT mice (open bars) and LPS CD40KO (filled bars) mice. Vertically striped bars, saline WT; horizontally striped bars, saline CD40KO. (C) Histologic sections of lung tissue obtained after 24 h following LPS or saline challenge in WT and CD40KO mice. (C1) LPS WT; (C2) LPS CD40KO; (C3) saline WT; (C4) saline CD40KO. Original magnification: x100. Sections shown are representatives of five sections of lung per mouse, from four mice in each group.

 
Histologic Evaluation of Lung Injury in Response to Intratracheal LPS
To confirm a lesser amplification of LPS-induced lung injury in CD40KO mice, we used a histologic evaluation of the lungs. As shown in Figure 1C, panel C1, the lungs in LPS WT mice stained with hematoxylin-eosin revealed widespread alveolar wall thickening caused by edema, severe hemorrhage in the interstitium and alveolus, and marked and diffuse interstitial infiltration with neutrophils and monocytes/macrophages, as well as significant airspace cellularity as reported previously (11). However, LPS CD40KO mice showed a significant decrease of interstitial infiltration and airspace cellularity, and their histological changes were minor compared with those in LPS WT mice, especially in the hemorrhaging of the interstitium and alveolus (Figure 1C, panel C2). Both WT mice (Figure 1C, panel C3) and CD40KO mice (Figure 1C, panel C4) injected with saline showed a normal lung histology. Quantal scoring (results expressed as mean ± SEM) of the severity of histologic lung injury (Table 1) showed that the lung-injury score was significantly higher (P < 0.05) in the LPS WT mice (226.0 ± 25.0) than in the LPS CD40KO mice (129.3 ± 6.5). These findings also support the idea that CD40 might be involved in the development and amplification of LPS-induced lung injury.


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  		TABLE 1. Changes in lung histology in WT and CD40KO mice

 
Cellular Characterization of Lung Inflammation in Response to Intratracheal LPS
As previously reported (11), the analysis for the cellular profile in BAL fluid supported the histologic findings. BAL was performed 24 h after LPS instillation intratracheally, and BAL fluid was collected. As shown in Figure 2A, the total cell number increased significantly in LPS WT mice, although it was clearly inhibited in LPS CD40KO mice (56.9 ± 8.7 x 104/ml and 20.7 ± 3.8 x 104/ml, respectively; P < 0.05), and most of the increased cells were neutrophils (51.4 ± 8.7 x 104/ml and 13.5 ± 2.2 x 104/ml, respectively; P < 0.05).



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  		Figure 2. Cellular characterization of lung inflammation in response to intratracheally injected LPS. (A) Inflammatory cell numbers in BAL fluid challenged by LPS or saline in WT and CD40KO mice. TCC, total cell counts; Neutro, neutrophils; Lym, lymphocytes; Mono/Macro, monocytes/macrophages; Eo, eosinophils. Open bars, LPS WT; filled bars, LPS CD40KO; vertically striped bars, saline WT; horizontally striped bars, saline CD40KO. Data represent the mean ± SEM for groups of six mice. *Significant differences (P < 0.05) in TCC or Neutro between LPS WT and LPS CD40KO mice. (B) Peripheral blood cell populations of WT and CD40KO mice challenged intratracheally by LPS or saline. Data represent the mean ± SEM for groups of four mice. No significant difference in Neutro was observed between WT and CD40KO mice challenged by LPS.

 
As previously reported, the influx of inflammatory cells into the tissue after LPS challenge is partly related to the content of cells in the peripheral circulation (16). In the absence of a CD40–CD154 interaction such as hyper IgM syndrome and CD40- or CD154-deficient mice, neutropenia or a decreased number of neutrophils has been observed (7, 16–18). Therefore, we investigated cell counts and conducted a differential analysis of the peripheral blood (Figure 2B). Although the number of neutrophils in blood was significantly low in CD40KO mice, an increased number of them was observed in response to LPS challenge, but the difference was not significant compared with LPS WT mice (31.4 ± 2.6% and 37.4 ± 4.0%, respectively; P = 0.22) (Figure 2B). Taken together with a previous study (16), CD40KO mice have been shown to possess sufficient hematopoietic capability in response to LPS stimulation. In addition, there might be other mechanisms that operate to diminish the recruitment of neutrophils into the lung tissue.

Evaluation of Proinflammatory Cytokines and Chemokine in LPS-Induced Lung Injury
The decreased response to LPS in CD40KO mice might be attributed to a decline in the release of systemically active proinflammatory cytokines, chemokines, MMPs, and reactive oxygen and nitrogen intermediates that are produced in inflammatory lesions and play important roles in regulating inflammatory cell recruitment into acute injury sites. These mediators may be produced locally in the lung by inflammatory cells, lung epithelial cells, or fibroblasts (1). We then examined the production of inflammatory mediators following an intratracheal challenge with LPS. Figure 3 illustrates TNF-{alpha}, IL-1ß, and MIP-2 levels in the serum and BAL fluid as representatives of proinflammatory cytokines and chemokines. With an intratracheal LPS challenge, TNF-{alpha}, IL-1ß, and MIP-2 levels in BAL fluid from WT mice were significantly elevated compared with those from CD40KO mice (TNF-{alpha}: LPS WT and LPS CD40KO, 732 ± 95 pg/ml and 386 ± 73 pg/ml, respectively; P < 0.05) (IL-1ß: 101 ± 17 pg/ml and 60.8 ± 10 pg/ml, respectively; P < 0.05) (MIP-2: 517 ± 94 pg/ml and 290 ± 69 pg/ml, respectively; P < 0.05) (Figures 3A, 3C, and 3E). In contrast, TNF-{alpha}, IL-1ß, and MIP-2 levels in serum were low and showed no statistical differences among experimental groups (Figures 3B, 3D, and 3F).



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  		Figure 3. Proinflammatory cytokines and chemokines in lung inflammation in response to intratracheal LPS. TNF-{alpha} (top panels), IL-1ß (middle panels), and MIP-2 (bottom panels) in BAL fluid (left panels) and serum (right panels) in WT and CD40KO mice challenged intratracheally by LPS or saline. Data represent the mean ± SEM for groups of five mice. *Significant differences (P<0.05) between LPS WT (open bars) and LPS CD40KO (filled bars) mice. Vertically striped bars, saline WT; horizontally striped bars, saline CD40KO.

 
Evaluation of Toxic and Destructive Mediators in Lung Injury by LPS
We next evaluated the role of MMPs in BAL fluids. BAL fluid from LPS WT mice showed an intensive band of gelatinolytic activity at 92 kD corresponding to proMMP-9 as well as several higher molecular weight bands of gelatinolytic activity, presumably representing lipocalin-associated latent forms of MMP-9, specific to neutrophils, and/or dimers of MMP-9 (Figure 4A1) (19). On the other hand, BAL fluid from LPS CD40KO mice showed significantly weak MMP-9 gelatinolytic activities compared with those from LPS WT mice (intensity value: 1120 ± 239 and 2699 ± 318, respectively; P < 0.05) (Figure 4A2). A very faint 72-kD band, probably representing MMP-2, was also present in BAL fluid. The culture supernatant of U937 cells, a human monocyte-like cell line, was used as a positive control (14).



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  		Figure 4. MMPs activity and nitrotyrosine staining in WT mice and CD40KO mice challenged intratracheally by LPS or saline. (A1) Estimation of MMP activity by zymography in BAL fluid of WT and CD40KO mice challenged intratracheally by LPS. Aliquots of BAL fluid were analyzed under nonreducing conditions by gelatin zymography. Intensive band of gelatinolytic activity at 92 kD corresponds to MMP-9. Culture supernatant of U937, a human monocyte-like cell line, was used as positive control. (A2) Measurement of MMP-9 activity. Intensities of MMP-9 bands were estimated by the NIH image 1.61 program. Data represent the mean ± SD for groups of six mice. Open bars, LPS WT; filled bars, LPS CD40KO. (B) Immunostaining for nitrotyrosine in lung tissues of WT and CD40KO mice administered with LPS or saline. (B1) LPS WT (original magnification: x100; inserted panel: x400); (B2) LPS CD40KO (x100; inserted panel: x400); (B3) saline WT (x200); (B4) saline CD40KO (x200). Result is representative of four experiments.

 
Figure 4B showed immunohistochemical staining for tyrosine nitration in the lungs. Although the expression of nitrotyrosine could be weakly detected in airway epithelial cells of both LPS WT and CD40KO mice, the lung tissues showed widespread nitrotyrosine staining in the inflammatory lesions of LPS WT mice (Figure 4B1), but not of LPS CD40KO mice (Figure 4B2). The lung tissues of control groups evidenced little expression of nitrotyrosine (Figure 4B3 and 4B4; saline WT mice and saline CD40KO mice, respectively). We also examined nitrotyrosine levels by Western blotting. Whole lungs were homogenized and proteins were immunoblotted after electrophoresis with the same antinitrotyrosine antibody as in the immunochemistry experiments. Several bands were detected in unchallenged animals. After LPS challenge and in both WT and CD40KO mice, some bands increased in density whereas others remained constant. No difference was found between wild-type and CD40KO mice in the pattern of nitration. A representative Western blotting obtained was shown in Figure 5. Compared with the immunohistochemistry, we did find any marked difference between wild-type and CD40KO mice.



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  		Figure 5. Detection of nitrotyrosine by Western blotting in whole lung homogenates from WT (lanes 1 and 2) and CD40KO mice (lanes 3 and 4) under control (lanes 1 and 3) or LPS-challenged conditions (lanes 2 and 4). One hundred fifty micrograms of protein extract was separated on 10% SDS-PAGE gel and transferred onto PVDF membranes. Result is representative of four experiments.

 
These findings suggested that the decreased recruitment of neutrophils into the lung tissue in LPS CD40KO mice might be explained by the decreased expression of candidate molecules for ALI, which included TNF-{alpha}, IL-1ß, MIP-2, MMP-9, and nitrogen intermediates.

Evaluation of LPS Receptors in LPS-Induced Lung Injury
We further evaluated whether the decrease in LPS-induced lung injury in CD40KO mice might be due to the lack of expression of LPS receptors, including CD14 and SRA. Figures 6A–6H show the immunohistochemical staining of lung tissues with anti-CD14 antibody and anti-SRA antibody. Expressions of CD14 and SRA could be detected in alveolar macrophages both in WT and CD40KO mice with intratracheal LPS administration (SRA, Figures 6A and 6C; CD14, Figures 6E and 6G). The expression of CD14 could barely be detected in saline-treated groups (SRA, Figures 6B and 6D; CD14, Figures 6F and 6H). To quantify the expression of CD14 and SRA protein, whole lung homogenates of WT and CD40KO mice were analyzed by Western blotting with the same anti-CD14 and anti-SRA antibodies as in the immunochemistry experiments. Quantification of these blots for CD14 and SRA was presented in Figures 6I and 6J, revealing no significant differences between the LPS WT and LPS CD40KO mice (P = 0.759 and P = 0.7824, respectively). These results showed that there was no difference in LPS receptor expression between WT and CD40KO mice.



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  		Figure 6. Representative immunohistochemical staining of LPS receptors. Immunostaining for SRA and CD14 in lung tissues of WT and CD40KO mice administered LPS or saline. (A–D) SRA immunostaining; (E–H) CD14 immunostaining. (A, E) LPS WT; (B, F) saline WT; (C, G) LPS CD40KO; (D, H) saline CD40KO. (I, J) Detection of CD14 and SRA by Western blotting in whole lung homogenates, respectively, from WT (lanes a and b) and CD40KO mice (lanes c and d) under control (lanes a and c) or LPS-challenged conditions (lanes b and d). One hundred fifty micrograms of protein extract was separated on 10% SDS-PAGE gels and transferred onto PVDF membranes. Result is representative of four experiments.

 
Effect of CD40 in Activation of LPS-Stimulated Resident Alveolar Macrophages
To investigate whether the decreased LPS-induced lung injury in CD40KO mice would be due to a decrease in the initial inflammatory responses in resident cells of the lung, we isolated naive AM{phi} from mouse lungs and examined the functional ability of AM{phi} stimulated with LPS in vitro.

First, we evaluated CD40 expression on AM{phi} obtained from WT mice. Although weak expression of CD40 was detected in naive AM{phi} without LPS stimulation (Figure 7A1), its expression on AM{phi} was induced after stimulation with LPS in a dose-dependent manner (0, 0.1, 1.0, 10 µg/ml) (Figures 7A2–7A4). Furthermore, CD40 was very rapidly upregulated on LPS-stimulated AM{phi}, and 6 h after LPS-stimulation it revealed maximum expression (Figure 7A5). CD40 could not be detected on LPS-stimulated CD40KO AM{phi} (data not shown).



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  		Figure 7. CD40 and iNOS expressions in LPS-stimulated resident alveolar macrophages. WT and CD40KO AM{phi} (5 x 105 cells/ml) were incubated with or without 0.1, 1, or 10 µg/ml LPS, or 300 U/ml mIFN-{gamma} for 24 h. In separate experiments, AM{phi} were also incubated with 10 µg/ml LPS for different incubating times, such as 0, 6, or 24 h. (A) Immunohistochemical expression of CD40 on WT AM{phi} to LPS stimulation. (A1) Vehicle for 24 h; (A2) LPS 0.1 µg/ml for 24 h; (A3) LPS 1 µg/ml for 24 h; (A4) LPS 10 µg/ml for 24 h; (A5) LPS 10 µg/ml for 6 h. (B) Immunohistochemical expression of iNOS in WT and CD40KO AM{phi} to LPS or IFN-{gamma} stimulation. (B1a–B4a) WT AM{phi}; (B1b–B4b) CD40KO AM{phi}. (B1a and B1b) Vehicle; (B2a and B2b) LPS 1 µg/ml; (B3a and B3b) LPS 10 µg/ml; (B4a and B4b) IFN-{gamma} 300 U/ml. Insets are magnified views. (C) Nitrite concentration in the culture supernatants of WT and CD40KO mice to LPS stimulation. Results represent the mean ± SD of three independent experiments. *Significant differences (P < 0.05) between LPS WT AM{phi} and LPS CD40KO AM{phi}.

 
When we investigated iNOS expression in AM{phi}, we found it to be undetectable in both WT (a) and CD40KO mice (b) without LPS stimulation (Figures 7B1a and 7B1b). Although the expression of iNOS was induced in AM{phi} from WT mice after stimulation with LPS (1 and 10 µg/ml, and percentage of positive cells; 1.2%, 2.8%, respectively) (Figures 7B2a and B3a), it was not observed in AM{phi} from CD40KO mice (Figures 7B2b and 7B3b). However, when AM{phi} was stimulated with IFN-{gamma}, the expression of iNOS was detected in AM{phi} from both WT and CD40KO mice (percentage of positive cells: 2.2% and 5.1%, respectively, Figures 7B4a and 7B4b). To confirm the different expression of iNOS in AM{phi}, we assessed the nitrite concentration in the culture supernatants of AM{phi}. Shown in Figure 7C, low levels of nitrite could be detected in the culture supernatants without LPS stimulation. Nitrite was significantly produced in the culture supernatants of AM{phi} from WT mice under stimulation with LPS. In contrast, no increase in nitrite production was observed in the culture supernatants of AM{phi} from CD40KO mice under stimulation with LPS (WT AM{phi}, 23.0 ± 3.4 µM versus CD40KO AM{phi}, 5.16 ± 3.2 µM; P < 0.05). The same level of nitrite production was observed in culture supernatants under stimulation with IFN-{gamma} (data not shown). These findings indicated that AM{phi} from CD40KO was impaired in its ability to induce iNOS expression and NO production even at high doses of LPS stimulation (10 µg/ml), and that CD40 on AM{phi} might be essential to the signal pathway for the induction of iNOS expression and NO production in AM{phi} by LPS stimulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated in our LPS-induced ALI model that the histologic degree of injury and the level of inflammatory mediators were significantly reduced in mice deficient in CD40. Furthermore, AM{phi} purified from CD40KO mice could not induce iNOS expression in response to LPS, although iNOS expression in AM{phi} from WT mice was induced in response to LPS independently of the CD40–CD154 interaction. These results indicated that activation of AM{phi} through CD40 might be involved not only in the amplification by interaction with CD154 but also in the development of acute lung injury by CD40 itself in LPS-induced lung inflammation. We first demonstrated that a loss of CD40 surface expression was enough to interrupt the production of iNOS in response to LPS by AM{phi}.

To clarify the inflammatory response in the lung, we used a single, intratracheal administration of LPS. This method resulted not only in the local release of inflammatory mediators from lung constituents such as AM{phi}, epithelial cells, and endothelial cells, but also in the recruitment of inflammatory cells into the alveolar space. An LPS-induced ALI model was known to be both macrophage- and neutrophil-dependent (20). Cumulative evidence supports the important role of neutrophils in the pathogenesis of ALI and ARDS. Neutropenia or a decrease in the neutrophil population is often observed in the absence of a CD40–CD154 interaction (16–18). However, we demonstrated that CD40KO mice increased the number of neutrophils in peripheral blood in response to LPS, and that the number was not statistically different from that observed in WT mice. These findings suggested that CD40KO mice appeared to have sufficient hematopoietic capability in response to LPS, and that neutropenia in the steady state of CD40KO mice might not be the cause of the decreased recruitment of neutrophils into the lung tissue. Several mediators are reported to recruit neutrophils into inflammatory sites. Next, we examined the amounts of MIP-2 and TNF-{alpha} in BAL fluids. MIP-2, a functional analog of human IL-8, is an important mediator in the recruitment of neutrophils (21). TNF-{alpha} acts locally to stimulate chemotaxis and activate neutrophils (22). We showed that the production of TNF-{alpha} and MIP-2 from LPS-stimulated CD40KO mice was attenuated in comparison with that in LPS-stimulated WT mice. Low concentrations of MIP-2 and TNF-{alpha} in the BAL fluids of CD40KO mice might result in a decreased recruitment of neutrophils to some extent.

To evaluate the AM{phi}-dependent portion of the LPS-induced ALI focusing on activation through CD40, we first examined the expression of CD40 on AM{phi} purified from WT mice. Without LPS stimulation, naive AM{phi} expressed CD40 weakly, as we reported previously (5), and its expression was induced very rapidly after LPS stimulation in a dose-dependent manner (Figure 7A1). CD154 expression has been detected on activated T lymphocytes (6) and mast cells (23), and we also detected its expression in the tissue of injured lungs by in situ hybridization (data not shown).

iNOS expression, NO production, the release of systemically active proinflammatory cytokines, chemokines, and MMPs have been known to play an important role in regulating the inflammation in acute lung injury. Many kinds of these inflammatory mediators are produced through CD40–CD154 interaction (6). High concentrations of TNF-{alpha}, IL-1ß, and IL-8 could be detected in the BAL fluids of patients with ARDS (24). We showed that the production of TNF-{alpha}, IL-1ß, and MIP-2 from LPS-stimulated CD40KO mice was attenuated in comparison with that in LPS-stimulated WT mice. Furthermore, the aberrant expression of MMPs could cause tissue damage and induce lung injury. These MMPs are produced during macrophage-dependent lung injury, and AM{phi} is their likely source (3, 25). We demonstrated that the gelatinolytic activity of MMP-9 in CD40KO mice was attenuated compared with that in WT mice, and this might suggest that the signal through CD40 in the lung were associated with the amplification of ALI. A CD40–CD154 interaction is primarily implicated in the establishment of humoral immunity, especially T cell–dependent humoral immune responses (16–18). In recent years, however, it has become clear that CD40 is expressed much more broadly, not only on monocytes/macrophages and dendritic cells but also on mesenchymal cells, such as endothelial cells and fibroblasts (6). Engagement of CD40 on these cells has been reported to lead to upregulation of costimulatory and cell adhesion molecules, as well as secretion of proinflammatory cytokines and effects on cellular proliferation (6). Endothelial cells were important in ALI and reported to play a critical role in vascular leakage (26). These constituents of the lung might also produce the inflammatory mediators and play some roles in amplifying inflammation in the lung.

To elucidate the precise role of the activation of AM{phi} through a CD40 signal in LPS-induced ALI, we examined the expression of iNOS and NO production on purified AM{phi} in vitro. Unlike in the case of AM{phi} from WT mice, no expression of iNOS or NO production could be detected in AM{phi} without CD40 expression, whereas such expression was equally detected both in WT and CD40KO mice by IFN-{gamma} stimulation. These results suggested that a CD40 signal for the activation of AM{phi} was involved in the development of LPS-induced ALI. Previous studies have demonstrated that NO-dependent pathways were important in the lungs of patients before the onset and during the course of ARDS (27), and peroxynitrite has been proposed to play a key role in mediating NO-related cellular injury (28). Moreover, the experimental lung injury model using iNOS-deficient mice indicated the attenuation of acute injury of the lung stimulated by LPS (29). These findings may support our results that the attenuation of iNOS expression and NO production in AM{phi} from CD40KO mice resulted in a decrease in acute lung injury induced by LPS. It has been reported that the TNF receptor–associated factor (TRAF) 6 is associated directly with the cytoplasmic tail of CD40, and could be involved in CD40-mediated activation of nuclear factor-{kappa}B. A finding similar to ours was reported in TRAF6–/–bone marrow macrophages. Although these macrophages were impaired in their ability to induce iNOS even under high LPS stimulation, the combination of TNF-{alpha} plus IFN-{gamma} activated the iNOS in them. An overexpression of dominant-negative TRAF6 impairs a toll-like receptor (TLR) 4-induced nuclear factor-{kappa}B activity (30). Moreover, Lomaga and coworkers demonstrated directly that TRAF6 is crucial not only to CD40 signaling but also to LPS signaling using TRAF6 knockout (TRAF6–/–) mice (31). One of the mechanisms of the attenuated response to LPS in the ALI model of CD40KO mice seems to be involved in the LPS signaling pathway, and TRAF6 could well play some role downstream of the signal pathways from both CD40 and CD14/TLR4. However, no information is available to indicate the role of the TRAF6 in iNOS expression and NO production in AM{phi} from CD40KO mice.

The LPS-induced activation of inflammatory cells could be attributed to the signal through several LPS receptors, including CD14 and SRA, because mice lacking these LPS receptors proved highly resistant to shock induced by LPS (32, 33). To exclude the possibility that the expressions of LPS receptors were downregulated in mice deficient in CD40, we showed that the expression of CD14 and SRA could be similarly detected in both WT and CD40KO mice in response to LPS, thus indicating that the expression of CD14 and SRA might not cause the inhibition of LPS-induced ALI in CD40KO mice. However, we could not rule out other possibilities, e.g., the role of a CD14-independent pathway of macrophage activation induced by LPS stimulation, such as heat shock protein 70, heat shock protein 90, chemokine receptor (CXCR) 4, growth differentiation factor 5 (34), or TLR4.

In summary, we have provided experimental evidence on the crucial roles of CD40 in both the macrophage- and neutrophil-dependent portions of LPS-induced acute lung inflammatory responses as well as in both its development and amplification. We further demonstrated that CD40 might be an essential molecule for iNOS expression in AM{phi} induced by LPS. Our study further suggested that the functional blockade of CD40 might yield one of the targets for clinical treatment of LPS-induced ALI and ARDS.


   Footnotes
 
* These authors contributed equally to this work. Back

Received in original form May 16, 2003

Received in final form December 5, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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