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Published ahead of print on March 14, 2003, doi:10.1165/rcmb.2002-0132OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 252-258, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0132OC

Effect of CD14 Blockade on Endotoxin-Induced Acute Lung Injury in Mice

Sadatomo Tasaka, Akitoshi Ishizaka, Wakako Yamada, Mie Shimizu, Hidefumi Koh, Naoki Hasegawa, Yoshiyuki Adachi and Kazuhiro Yamaguchi

Department of Medicine, Keio University School of Medicine; Department of Laboratory Medicine, Tokyo Electric Power Company Hospital; and Laboratory of Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Science, Tokyo, Japan

Address correspondence to: Akitoshi Ishizaka, M.D., Department of Laboratory Medicine, Tokyo Electric Power Company Hospital, Shinjuku-ku, Tokyo 160-1100, Japan. E-mail: ishiz{at}attglobal.net


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD14 functions as a cell surface receptor for endotoxin (lipopolysaccharide [LPS]) and is thought to have an essential role in innate immune responses to infection. Previous studies have revealed attenuation of the systemic response after sepsis by blocking CD14. In this study, we tested the hypothesis that CD14 blockade protects against inflammatory responses associated with LPS pneumonia. We examined the effect of an anti-murine CD14 monoclonal antibody (4C1) on the development of acute lung injury induced by intratracheal LPS in mice. We also measured the production of cytokines (tumor necrosis factor-{alpha}, interleukin-6, and macrophage inflammatory protein-2) and nitric oxide by murine peritoneal macrophages exposed to LPS in vitro. Nuclear factor (NF)-{kappa}B translocation was evaluated in nuclear extracts from lung homogenates. 4C1 significantly attenuated pulmonary edema and neutrophil emigration after LPS administration. The production of cytokines and nitric oxide by LPS-stimulated macrophages was significantly decreased by 4C1 treatment. NF-{kappa}B translocation induced by LPS instillation was also suppressed by 4C1. These results suggest that blockade of CD14 might attenuate acute lung injury after intratracheal instillation of LPS through the suppression of NF-{kappa}B translocation. The inhibitory effect of CD14 blockade on cytokine production and nitric oxide release of macrophages might contribute to the attenuation of lung injury.

Abbreviations: acute respiratory distress syndrome, ARDS • bronchoalveolar lavage, BAL • PBS-T containing 0.5% bovine serum albumin, BPBS-T • enzyme-linked immunosorbent assay, ELISA • electrophoretic mobility shift assay, EMSA • extravascular albumin, EVA • interleukin, IL • LPS-binding protein, LBP • lipopolysaccharide, LPS • macrophage inflammatory protein, MIP • nuclear factor-{kappa}B, NF-{kappa}B • phosphate-buffered saline, PBS • PBS containing 0.05% Tween 20 • PBS-T • red blood cells, RBC • reticuloendothelial system, RES • tumor necrosis factor-{alpha}, TNF-{alpha} • white blood cells, WBC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The acute respiratory distress syndrome (ARDS) is a process of acute inflammatory lung injury resulting from a variety of predisposing conditions including severe pneumonia, sepsis, massive transfusion, and trauma (1, 2). Endotoxin, lipopolysaccharide (LPS), a major component of the cell wall of gram-negative bacteria, plays an important role in the pathogenesis of ARDS (3). Recognition of LPS by the host initiates several intracellular signal transduction pathways, which in turn result in cell activation and production of a variety of endogenous mediators, including proinflammatory cytokines, adhesion molecules, and nitric oxide (4).

CD14 is a myeloid cell differentiation antigen expressed primarily on monocytes, macrophages, and neutrophils (5). Recent work has demonstrated that recognition of LPS by host cells is principally mediated by either a membrane-bound or a soluble form of CD14 (6–8). Binding of LPS to CD14 requires a serum factor, LPS-binding protein (LBP) (9). Binding of the LPS–LBP complexes to CD14 triggers the activation of various signal transducers including nuclear factor (NF)-{kappa}B (10). NF-{kappa}B is a dimeric transcription factor that is present in the cytosol in an inactive form bound to its inhibitory protein, I-{kappa}B. When activated by a variety of stimuli, NF-{kappa}B translocates to the nucleus, where it binds to DNA and promotes the production of many molecules, such as proinflammatory cytokines including interleukin (IL)-1 and tumor necrosis factor (TNF)-{alpha} (10). This pathway of macrophage activation is thought to be a major contributor to the development of LPS-induced acute lung injury.

In most cases, patients with severe pneumonia are treated with antibiotics with a broad spectrum, which is usually effective against gram-negative bacteria. It has been shown that antibiotic therapy provides no benefit to the outcome of ARDS subsequent to severe pneumonia, even if the antibiotics administered are effective against the pathogenic bacteria (11). We think this finding means that gram-negative bacteria contribute to the development of ARDS by releasing endotoxin even after they are destroyed by antibiotics. Therefore, we hypothesized that blockade of CD14 is important to prevent the progress of ARDS.

It has been reported that anti-CD14 mAb protects against organ injury in a rabbit model of endotoxic shock (12). CD14-deficient mice were found to have strong resistance against intraperitoneal LPS and revealed to have decreased cytokine production in response to LPS (13). However, there has been no report concerning the effect of CD14 blockade on the inflammatory response to an insult administered intratracheally, except for a recent report by Frevert and colleagues, who described that CD14 blockade significantly improved systemic physiological responses in rabbits with Escherichia coli pneumonia, but impairment of alveolar oxygenation by blocking CD14 was observed as well (14).

The goal of this series of experiments was to determine the effects of CD14 blockade on the development of acute lung injury after intratracheal instillation of LPS using a murine model. The effect of CD14 blockade on NF-{kappa}B translocation was also determined using electrophoretic mobility shift assay (EMSA). In addition, we examined whether treatment with an anti-CD14 mAb, 4C1, makes changes in the production of cytokines (TNF-{alpha}, IL-6, and macrophage inflammatory protein [MIP]-2) and nitric oxide by murine peritoneal macrophages exposed to LPS in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
A monoclonal antibody against murine CD14 was prepared from the serum-free culture supernatant of a hybridoma clone, 4C1. The antibody 4C1, was purified by HiTrap protein G column chromatography (Amersham Biosciences, Buckinghamshire, UK). The eluted antibody fraction was dialysed against phosphate-buffered saline (PBS), and endotoxin contamination was confirmed to be under the detection limit of the limulus test (Seikagaku Co., Tokyo, Japan). 4C1 has been revealed to block LPS binding to cells expressing CD14 (15). E. coli endotoxin (serotype B:55) was purchased from Sigma Chemicals (St. Louis, MO).

Murine Model of Acute Lung Injury
To determine the effect of anti-CD14 mAb on LPS-induced acute lung injury, four groups of C57BL/6 mice (8–12 wk old; CLEA Japan, Tokyo, Japan) were studied. Group 1 was given an intravenous injection of isotype-matched control mAb (2E6; 4 mg/kg) 15 min before instillation of PBS (50 µl); Group 2 received anti-CD14 mAb (4C1; 4 mg/kg) before PBS instillation; Group 3 received isotype-matched control mAb followed by the instillation of LPS (10 mg/kg in 50 µl/22 g PBS); Group 4 was treated with 4C1 before LPS challenge.

Mice were anesthetized using ketamine hydrochloride (80–100 mg/kg intramuscularly) and acepromazine maleate (5–10 mg/kg intramuscularly). Anesthetized mice received an intravenous injection of 125I-labeled human albumin (0.1 mCi/mouse) to measure edema, together with either 4C1 or isotype-matched control antibody 15 min before instillation. The trachea was exposed through a midline incision in the neck, and a 24-gauge catheter was inserted. Then, the animals received intratracheal administration of either PBS or LPS. Two minutes before the end of the study, 51Cr-labeled red blood cells (RBC; 0.1 mCi/mouse) were injected to measure the intravascular blood volume. After 6 h, the lungs were removed and fixed using intratracheal instillation of 6% glutaraldehyde at 22 cm H2O. Blood samples were obtained from the inferior vena cava. White blood cells (WBC) were counted using a hemocytometer, and differentials were determined using blood smears stained with Leukostat (Fisher Scientific, Pittsburgh, PA).

Edema in the lungs was evaluated by quantitating the accumulation of extravascular albumin (EVA) as previously described (16). The isotope-specific radioactivity of excised, fixed lungs as well as samples of blood and plasma was measured (ARC-300; Aloka, Tokyo, Japan) before sectioning the lungs for morphometric analysis. Blood and plasma volumes of the lungs were determined from 125I-albumin and 51Cr-RBC counts, respectively. Pulmonary edema (EVA in the lung) was calculated as total lung albumin content minus intravascular lung albumin content. Intravascular lung albumin content was calculated from pulmonary blood volume and hematocrit for each animal.

Pulmonary neutrophils were quantified by morphometric analysis in histologic sections (17). Paraffin-embedded 5-µm sections of lungs were cut and stained with hematoxylin and eosin. Neutrophil emigration was quantitated by counting the number of neutrophils in 200 randomly selected alveoli and expressed as the number of neutrophils per 100 alveoli.

Nuclear Protein Extraction from Lungs
Mice were anesthetized as above and received intratracheal instillation of PBS or LPS (10 mg/kg) 15 min after pretreatment with an intravenous injection of either 4C1 or control Ab. After 3 h, the lungs were removed and frozen immediately in liquid nitrogen. Nuclear extracts were prepared as previously described (18). Briefly, the isolated lungs were homogenized in 3 ml of ice-cold Buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF), with a 0.1% volume of Nonidet P-40 and protease inhibitor cocktail (1 mg/ml leupeptin, 1 mg/ml aprotinin, 10 mg/ml soy bean trypsin inhibitor, 1 mg/ml pepstain). Following 10-min incubation on ice, the homogenates were centrifuged at 850 x g for 10 min at 4°C. The pellets were resuspended in 3 ml of Buffer A and centrifuged at 1,400 x g for 10 min at 4°C. The crude nuclear pellets were resuspended in 50 ml of Buffer B (20 mM HEPES, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 25% vol/vol glycerol, 0.5 mM DTT, 0.5 mM PMSF) with protease inhibitor cocktail (as described above) and incubated for 30 min on ice. Nuclear extracts were recovered following centrifugation at 20,000 x g for 15 min at 4°C and stored at -80°C. Protein concentration of nuclear extracts was determined by bicinchoninic acid assay. The regression curve was obtained using bovine serum albumin as the standard.

EMSA
EMSA was performed following the manufacturer's protocol (Gel Shift Assay Systems; Promega, Madison, WI) with several modifications. The double strand NF-{kappa}B consensus oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') was end-labeled with 32P-ATP (3,000 Ci/mmol at 10 mCi/ml) using T4 polynucleotide kinase. Binding reactions (10 ml) containing 35 fmol/ml of oligonucleotide probe, 5 mg of nuclear extract and 5x binding buffer (10 mM Tris-HCl, 1 mM MgCl2, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 4% vol/vol glycerol, 0.5 mg poly dI-dC) were incubated for 20 min at room temperature. Following the binding procedure, 10-fold loading buffer (250 mM Tris-HCl, 0.2% bromophenol blue, 40% glycerol, 0.2% xylane cyanol) was added to stop the reaction. The reactions were subjected to non-denaturing 6% polyacrylamide gel electrophoresis (NOVEX, San Diego, CA) in 0.5x TBE buffer (45 mM Tris-boric acid, 1 mM EDTA-2H2O) at 65 V for 85 min. Gels were vacuum dried for 45 min and exposed to X-ray film (BMR; Kodak, Rochester, NY) at -70°C. Autoradiographic images were converted into numerical data by quantitative scanning laser densitometry.

Stimulation of Macrophages In Vitro
Mouse peritoneal macrophages were collected as previously described (19). Briefly, C57Bl/6 mice were injected intraperitoneally with 2 ml of 4% thioglycolate broth (Difco, Grayton, GA). Four days later, thioglycolate-induced peritoneal exudate cells were collected from the peritoneal cavity by washing twice with 5 ml of Hanks' Balanced Salt Solution (Sigma) containing heparin (5 U/ml). Isolated peritoneal cells were cultured for 2 h at a concentration of 2 x 106 cells per well in flat-bottom 24-well plates to make peritoneal macrophages adhere. Nonadherent cells were removed by flushing twice with RPMI1640 (Sigma), and adherent cells were used as peritoneal macrophages. The macrophages were pre-cultured for 10 min with 10 or 25 µg/ml of anti-murine CD14 mAb (4C1, rat IgG2b) or isotype control mAb (anti-TNP mAb, clone A8), then the cells were exposed to LPS and cultured for 24 h at 37°C. The neutralizing Ab was present for the entire culture period. Cultures were maintained in a 1-ml volume, and supernatants were assayed for TNF-{alpha}, IL-6, and MIP-2 by ELISA (PharMingen, San Diego, CA); nitric oxide concentration in the supernatant was also measured as described below.

ELISA for Cytokine Determination
Microtiter ELISA plates (NUNC) were coated with the capture antibody for TNF-{alpha} (rat IgG1; PharMingen), IL-6 (rat-IgG1; PharMingen), or MIP-2 (R&D Systems, Minneapolis, MN). The antibody was diluted in bicarbonate buffer (pH 9.5) to a final concentration of 0.25 µg/well, and 50 µl was added to each well. Plates were incubated at 4°C overnight, followed by washing three times with PBS containing 0.05% Tween 20 (PBS-T). Blocking was achieved by adding 100 µl/well of PBS-T containing 0.5% bovine serum albumin (BPBS-T) and then incubating the mixture at 37°C for 1 h. After three more wash cycles, rmTNF-{alpha} (PharMingen), rmIL-6 (PharMingen), MIP-2 (R&D Systems), and supernatant samples were added to the plate (50 µl/well) and left to incubate for 1 h at 37°C. After three washings with PBS-T and blocking with BPBS-T for 10 min, bound soluble cytokine was detected using a biotin-labeled anti–mTNF-{alpha} mAb (PharMingen), biotin-labeled anti–mIL-6 mAb (PharMingen), or biotin-labeled anti–MIP-2 mAb (R&D Systems). Antibodies were diluted in BPBS-T, and 50 µl/well was added for 1 h at 37°C. The plates were washed another three times, and 50 µl of 1:10,000 diluted streptavidin-conjugated horseradish peroxidase (Zymed, South San Francisco, CA) was added to each well. After incubation for 40 min and four final washes with PBS-T, the plates were developed using 50 µl/well of tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The reaction was stopped by the addition of 50 µl 1 N H3PO4/well and read using a microplate reader (MTP-32; Corona Electric, Katsuta, Japan) at 450 nm.

Nitric Oxide Production
Release of NO in the macrophage culture supernatant was measured by Griess assay (20). Briefly, 50 µl supernatant was reacted for 10 min at room temperature with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diaminedihydrochloride, 2.5% phosphoric acid). Optical density was measured at 550 nm. Nitrite content was quantified by comparison with a standard curve generated with sodium nitrite in the range of 0 to 100 µM.

Statistics
Data are presented as mean ± SEM. One-way ANOVA, Fisher's least-significant difference test, t test, and paired t test were used as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of CD14 Blockade on Development of Edema
To determine whether CD14 blockade affects the development of acute lung injury, pulmonary edema, which was represented as EVA, was evaluated (Figure 1). Animals treated with intratracheal LPS had significantly higher EVA than control mice that received PBS instillation (P < 0.01). Mice administered 4C1 before LPS revealed significantly decreased EVA as compared with those given control antibody and LPS (P < 0.05). There was no significant difference in EVA between the groups that received PBS instillation following intravenous administration of either 4C1 or control antibody.



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  		Figure 1. Edema formation 6 h after instillation. Mice received an intravenous injection of either PBS (open bars) or anti-CD14 mAb (closed bars). Regardless of 4C1 treatment, mice administered LPS revealed increased EVA (*P < 0.05). Blockade of CD14 decreased the edema formation after LPS instillation (#P < 0.05). n = 6 in each group.

 
Effect of Anti-CD14 mAb on Neutrophil Emigration
Morphometric quantitation of intra-alveolar neutrophils was performed to assess the effect of anti-CD14 mAb on neutrophil emigration after LPS instillation (Figure 2). Mice given intratracheal LPS had significantly increased neutrophil emigration compared with the groups that received PBS instillation (P < 0.01). Mice, which were administered 4C1 before LPS, revealed significantly decreased neutrophil emigration as compared with those given control antibody and LPS (P < 0.05). The number of emigrated neutrophils did not differ between the groups that received PBS instillation.



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  		Figure 2. Neutrophil emigration 6 h after instillation. Mice received an intravenous injection of either PBS (open bars) or anti-CD14 mAb (closed bars). Regardless of 4C1 treatment, mice administered LPS revealed increased neutrophil emigration (*P < 0.05). Blockade of CD14 decreased the neutrophil emigration after LPS instillation (#P < 0.05). n = 6 in each group.

 
Circulating WBC and Neutrophil Counts
Circulating WBC and neutrophil counts in blood samples obtained from the inferior vena cava are shown in Table 1. Both WBC and neutrophil counts were increased in mice administered intratracheal LPS compared with those treated with intratracheal PBS (P < 0.05). CD14 blockade made no significant difference in WBC and neutrophil counts among the groups.


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  		TABLE 1 Circulating WBC and neutrophil counts

 
Effect of Anti-CD14 mAb on NF-{kappa}B Translocation
NF-{kappa}B translocation after instillation of PBS or LPS was estimated using EMSA of whole lung nuclear extracts at 3 h (Figure 3). The mice were pretreated with either 4C1 or control Ab. In mice, which received control Ab before LPS instillation, NF-{kappa}B translocation was increased compared with that in those administered control Ab and PBS (P < 0.001). In animals that received LPS after 4C1 treatment, NF-{kappa}B translocation was significantly inhibited as compared with that in those treated with control Ab before LPS instillation (P < 0.001). No significant difference in NF-{kappa}B translocation was observed between the groups that received PBS instillation.



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  		Figure 3. NF-{kappa}B translocation 3 h after instillation. Mice received an intravenous injection of either PBS or anti-CD14 mAb before instillation of either PBS or LPS. Regardless of 4C1 treatment, mice administered LPS revealed increased NF-{kappa}B translocation after LPS instillation (*P < 0.01). Blockade of CD14 significantly decreased the NF-{kappa}B translocation after LPS instillation (#P < 0.05). n = 3 in each group.

 
Effect of Anti-CD14 mAb on LPS-Induced Cytokine Production
To validate the effect of anti-CD14 mAb (4C1) on LPS-induced macrophage activation, the macrophage production of inflammatory cytokines (TNF-{alpha}, IL-6, and MIP-2) was examined after exposure to various concentrations of E. coli LPS in vitro. The level of TNF-{alpha} in the culture supernatant was significantly decreased by pretreatment with anti-CD14 mAb when macrophages were stimulated with a lower concentration of LPS (Figure 4A). Pretreatment with 4C1 made no significant difference in TNF-{alpha} production after exposure to 10 ng/ml LPS or higher. LPS induced marked release of IL-6 from murine macrophages, and 4C1 treatment significantly attenuated IL-6 production after stimulation with 1.25 or 2.5 ng/ml LPS (Figure 4B). MIP-2 production was significantly increased after exposure to LPS (Figure 4C). Pretreatment with anti-CD14 mAb induced a significant decrease in MIP-2 production when macrophages were stimulated with a lower concentration of LPS, but 4C1 pretreatment made no difference in MIP-2 production when macrophages were exposed to a higher concentration of LPS. Isotype-matched control antibody had no inhibitory effect on the production of any cytokine.





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  		Figure 4. Effect of CD14 mAb on LPS-mediated cytokine synthesis in macrophages. Macrophages were pretreated for 10 min with 10 or 25 µg/ml of anti-murine CD14 mAb (4C1) or isotype control mAb, then the cells were exposed to various concentrations of LPS. After incubation for 24 h, cytokine concentrations of (A) TNF-{alpha}, (B) IL-6, and (C) MIP-2 in the culture supernatant were determined by ELISA. Statistical analysis was performed by Student's t test. *P < 0.05, **P < 0.01. Symbols in A: filled squares, medium; open squares, isotype control (10 µg/ml); open circles, 4C1 (10 µg/ml); filled circles, 4C1 (25 µg/ml). Symbols in B and C: filled bars, medium; open bars, isotype control (10 µg/ml); striped bars, 4C1 (10 µg/ml); open bars, 4C1 (25 µg/ml).

 
Effect of CD14 Blockade on Nitric Oxide Production
To determine whether CD14 blockade affects nitric oxide production by macrophages, murine macrophages were pretreated with 4C1 and then stimulated with various doses of E. coli LPS. Nitric oxide production by LPS-stimulated macrophages was diminished by antibody pretreatment with 4C1 (Figure 5). Isotype-matched control antibody had no significant effect on the production of nitric oxide by murine macrophages.



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  		Figure 5. Effect of CD14 mAb on LPS-mediated nitric oxide production in macrophages. Macrophages were pretreated for 10 min with 10 or 50 µg/ml of anti-murine CD14 mAb (4C1) or isotype control mAb, then the cells were exposed to various concentrations of LPS. After incubation for 24 h, nitric oxide concentration was determined by Griess assay. Statistical analysis was performed by Student's t test. * P < 0.05. Filled squares, medium; open circles, 4C1 (10 µg/ml); filled circles, 4C1 (50 µg/ml).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study examined the effect of CD14 blockade on the development of acute lung injury induced by intratracheal LPS and on macrophage function after inflammatory stimuli. Pulmonary edema and neutrophil emigration after LPS challenge were attenuated by blocking CD14 using an anti-CD14 mAb, 4C1. In addition, NF-{kappa}B translocation after LPS challenge was significantly inhibited by 4C1 treatment. Pretreatment of murine macrophages with 4C1 significantly decreased LPS-induced production of inflammatory cytokines. The macrophage production of nitric oxide induced by LPS was also suppressed by 4C1. These studies suggest that CD14 blockade may attenuate the development of acute lung injury and suppress the activation of macrophage function after LPS challenge.

Although a number of studies have examined the effect of CD14 blockade on inflammatory responses in vivo, most of them evaluated the responses against stimuli administered intravenously (12, 13, 21–23). Frevert and colleagues estimated the effect of CD14 blockade on the systemic and pulmonary responses in rabbits with E. coli pneumonia (14). Although they showed that pretreatment with anti-CD14 mAb protected rabbits against sustained hypotension and reduced their intravenous fluid requirement, they found no difference in the number of bronchoalveolar lavage (BAL) neutrophils between rabbits with and without mAb treatment (14). In contrast, we observed a decrease in neutrophils, which were emigrated in alveolar spaces, in animals treated with 4C1. We speculate that this discrepancy might be due to the difference in stimuli. Whereas Frevert and colleagues inoculated live E. coli into the lungs, we instilled E. coli LPS, a component of the bacterial cell wall, into the airway to cause lung injury. There have been two other reports showing that CD14 blockade exacerbated bacterial infection (24, 25). Neutrophils are responsible for both host defense against bacterial pathogens and tissue injury by neutrophil elastase and reactive oxygen species. We think that blockade of innate immune responses could result in impairment of optimal neutrophil recruitment during the initial steps of infection, leading to delayed bacterial clearance, especially when infection is not controlled by antibiotics. In contrast, when bacterial activity is controlled by antibiotic therapy, blocking the function of CD14 might decrease neutrophil-mediated lung injury rather than increase tissue damage by bacterial infection. As most of patients with acute lung injury are treated with antibiotics, we believe that the attenuation of neutrophil accumulation into the lungs could benefit patients by preventing tissue injury.

Previous studies revealed CD14 expression in the liver, especially on Kupffer cells, in response to various inflammatory stimuli (26). CD14 could contribute to increased production and release of inflammatory mediators from the liver, which might accelerate the development of lung injury. We previously showed that BCG-induced activation of the reticuloendothelial system (RES) enhances LPS-induced lung injury (27). Although we did not examine cytokine levels in the systemic circulation, the attenuated response of the RES might contribute to suppression of the local inflammatory process in the lungs. We thought that intravenous 4C1 might suppress the inflammatory response of the RES including Kupffer cells, leading to the attenuation of lung injury.

The results of this study indicate that an anti-CD14 mAb, 4C1, may work via suppressing NF-{kappa}B translocation. NF-{kappa}B can be activated by various stimuli including microbial products, and in turn regulates the inducible expression of many cytokines, chemokines, and adhesion molecules (10). We observed decreased NF-{kappa}B translocation after 4C1 treatment using lung homogenates, but it remains unclear which types of pulmonary cells are responsible for this change.

In the present study, we measured cytokine release from macrophages in vitro and showed that CD14 blockade also diminished the production of inflammatory cytokines. In contrast, Frevert and colleagues showed no significant difference in cytokine levels in BAL fluid, and speculated that the effect of CD14 blockade on pulmonary host defenses might not be the result of impaired production of these cytokines (14). Cytokines are known to potentiate the function of neutrophils and macrophages, resulting in activation of anti-microbial mechanisms, including generation of superoxide, reactive oxygen and nitrogen intermediates, or enhancement of phagocytosis, with an increased ability to kill microorganisms (28). We think it possible that CD14 blockade might attenuate LPS-induced lung injury by suppressing the production of inflammatory cytokines.

Frevert and colleagues showed that rabbits treated with anti-CD14 mAb had less accumulation of NO-derived products in plasma, and speculated that CD14 blockade might improved the hemodynamics after E. coli inoculation by decreasing nitric oxide production (14). In the present study, we observed decreased production of nitric oxide by LPS-stimulated macrophages, which is consistent with the results of the study by Frevert and colleagues. Although nitric oxide is a potent vasodilator causing hypotension during sepsis (29), nitric oxide is known to have both beneficial and adverse effects on pulmonary inflammation, probably depending on its concentration (30). It has been revealed that nitric oxide reduces neutrophil recruitment during endotoxemia (31) and that nitric oxide reacts with oxygen and superoxide to form nitrogen dioxide, a potent pulmonary irritant, and peroxynitrite, a cytotoxic oxidant, respectively (30). We think that, in this study, reduced production of nitric oxide might have contributed to the attenuated lung injury after intratracheal instillation of LPS.

There have been few reports describing the effect of CD14 blockade on pulmonary edema development. Leturcq and colleagues revealed that CD14 blockade induced a significant decrease in protein leakage into BAL fluid following intravenous LPS (24). In the present study, we showed a decrease in EVA after LPS instillation in mice treated with 4C1. We think that this coincidence could support the importance of CD14 in pulmonary endothelial damage after LPS challenge.

Although blocking CD14 decreased albumin leakage, attenuated neutrophil emigration into the lungs, and blunted the inflammatory response, blockade of CD14 did not return these values to baseline. Although the LPS concentration in alveolar lining fluid in the mouse study is not clear, the LPS concentrations used for our in vitro studies are not extremely high compared with previous studies (32, 33). Several investigators reported cytokine production by macrophages from CD14-deficient mice exposed to LPS (13, 32). Perera and coworkers described CD14-independent signaling pathways after LPS stimulation in murine macrophages (32), and it was also shown that differential expression of cytokines (TNF-{alpha}, IL-1) is induced by various concentrations of LPS in macrophages lacking in CD14 (33). We think that there might be CD14-independent mechanisms that contribute to the LPS-induced tissue injury and inflammatory response observed in the mice treated with anti-CD14 mAb.

A limitation of this study is that there is no evidence about the binding site of 4C1. Although initially discovered in myeloid-derived cells, CD14 is also expressed by murine airway epithelial cells (34). In addition, it has been revealed that soluble CD14 in serum functionally replaces membrane-bound CD14 in CD14-negative nonmyeloid cells, such as endothelial cells (35, 36). It has been shown that soluble CD14 is elevated in both serum and bronchoalveolar lavage fluid of patients with ARDS (37), and that intratracheal instillation of anti-CD14 mAb prevented neutrophil sequestration induced by the LPS/LBP complex (38). It remains to be determined whether 4C1 treatment attenuates lung injury by blocking membrane-bound CD14 that exists on alveolar macrophages and epithelial cells or soluble CD14 in serum and alveolar lining fluid.

In conclusion, the results described here might demonstrate the significance of blocking the LPS/CD14 pathway in an in vivo model of LPS-induced acute lung injury that is similar to the events that occur in humans with severe pneumonia or ARDS.

Received in original form June 24, 2002

Received in final form February 26, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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