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Pain Medicine  |   December 2003
Acid-induced Lung Injury: Role of Nuclear Factor-κB
Author Affiliations & Notes
  • Lilly Madjdpour
    *
  • Sita Kneller, M.D.
  • Christa Booy
  • Thomas Pasch, M.D.
    §
  • Ralph C. Schimmer, M.D.
  • Beatrice Beck-Schimmer, M.D.
    #
  • * Medical Student, † Resident, ‡ Technical Assistant, § Professor and Chairman, # Associate Professor, Institute of Anesthesiology. ∥ Associate Professor, Department of Surgery.
  • Received from the Institute of Anesthesiology, Institute of Physiology, and Department of Surgery, University of Zurich, Zurich, Switzerland.
Article Information
Pain Medicine
Pain Medicine   |   December 2003
Acid-induced Lung Injury: Role of Nuclear Factor-κB
Anesthesiology 12 2003, Vol.99, 1323-1332. doi:
Anesthesiology 12 2003, Vol.99, 1323-1332. doi:
ACID aspiration is a well-known clinical situation. First described in 1946, 1 aspiration of gastric contents during anesthesia is still a cause of acute lung injury despite prophylactic procedures and improvements in supportive care. 2 It is a complication associated with high morbidity and mortality rates. Many studies have already been performed to better define the acute inflammatory mechanisms of acid aspiration. 3–5 Most of these models, however, did not investigate transcriptional mechanisms of acid-induced lung inflammation.
Transcription factors are expressed at the beginning of the inflammatory cascade. They are thus potential targets for blocking studies with a broad spectrum of possibilities for attenuating effects. Nuclear factor-κB (NF-κB) is one such critical transcription factor required for the expression of various proinflammatory molecules that play a crucial role in the pathogenesis of acute lung inflammation. 6 Some of the most prominent among these are the adhesion molecule intercellular adhesion molecule-1 (ICAM-1), the cytokine tumor necrosis factor-α (TNF-α), and the chemokines monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), and macrophage inflammatory protein-2 (MIP-2). They all play central roles in the recruitment of inflammatory cells in the lung. 7–10 NF-κB can be activated in cells by a variety of stimuli, including bacterial endotoxin, cytokines, mitogens, viral proteins, ionizing radiation, ultraviolet light, and certain chemical agents. 11 In previous studies, the activation of NF-κB has been linked to acute lung injury and has been shown to be a proximal step in the initiation of neutrophilic inflammation in animal models. 12,13 
Distal airway epithelial cells (alveolar epithelial cells [AECs]) are vital for the maintenance of the pulmonary air–blood barrier. Recent evidence suggests that airway epithelial cells may also act as immune effector cells in response to noxious endogenous or exogenous stimuli. Several studies have shown that AECs express and secrete various immune molecules, such as adhesion molecules, cytokines, and chemokines. 14–16 Through the expression and production of these inflammatory mediators, the airway epithelium is thought to play an important role in the initiation and exacerbation of inflammatory response within the airway.
The purpose of this study was to investigate acid-induced cellular and tissue lung injury in rat lungs and to analyze the time-course pattern of NF-κB activation and the expression of some of its gene products, such as ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2. Our research question furthermore was whether the expression of NF-κB could be reduced or inhibited and whether this could lead to an attenuation of the lung injury at the same time with a new administration form for the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), namely by intratracheal administration, a mode of administration with most likely fewer systemic effects than intravenous or intraperitoneal administration.
The hypothesis was tested whether biphasic cellular events in acid aspiration, as known from earlier studies, are also reflected by biphasic expression patterns of inflammatory mediators. 3 In addition, it was postulated that an intratracheal application of the NF-κB would attenuate lung injury in this model.
Materials and Methods
L2 Cells
L2 cells (CCL-149) were purchased from American Type Culture Collection (Rockville, MD). The L2 cell line was derived through clonal isolation after primary plating of a cell population from enzymatically dispersed adult rat lung. The cells have been shown to have the characteristics of rat alveolar epithelial type II cells. 17 L2 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Basel, Switzerland), which was supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, and 1% HEPES buffer. Using 35 × 10 mm plates (Corning, Corning, NY), L2 cells were grown until confluence.
Cytotoxicity Assay
Determination of cytotoxicity was performed with a chromium 51 assay. 18 L2 cells were placed into 96-well plates (NUNC Products; Life Technologies) and incubated at 37°C in 5% CO2. Twelve hours later, 0.8 μCi Na2[51Cr]O4(Amersham Pharmacia Biotech, Dubendorf, Switzerland) was added to each well and incubated for an additional 24 h. Monolayers were washed two times with medium, and experiments were started. In a first set of assays, cells were stimulated for 24 h with different pH values (7.4, 6, 5, 4, 3, and 2) by adding HCl to normal medium. One hundred–microliter aliquots of supernatants of stimulated cells were removed, and radioactivity was measured in a scintillation counter. Some wells were lysed with 0.1 ml 1% Triton X-100 to determine the total counts per minute initially bound to the cells at the beginning of the experiment. Cytotoxicity was calculated as the ratio of (A − B)/(C − B) × 100, in which A represents the release (counts per minute) from HCl-exposed cells and B is the counts per minute released from the control (unexposed cells, medium without HCl with pH = 7.6). C is the total counts per minute per well. To further compare the effects of pH values of 5 and 6, time courses over 2, 4, and 6 h were performed at these pH values and cytotoxicity was determined.
In vitro  Stimulation with HCl
A volume of 0.1 n endotoxin-free HCl (Sigma, Buchs, Switzerland) was added to Dulbecco's modified Eagle's medium and 1% fetal bovine serum with a pH value of 7.6, until a pH of 6 was reached. The mixture was then used for stimulation of confluent monolayers of L2 cells. The pH was controlled just before application. Cells were stimulated for 0.5, 1, 2, 4, 6, 8, and 24 h. Control cells were exposed to medium with a pH value of 7.4. RNA extraction was performed followed by reverse transcriptase–polymerase chain reaction.
Animals
Male Sprague–Dawley rats weighing 250–300 g were housed in individual isolator cages within the Animal Care Facilities at the University of Zurich. All animal experiments and animal care were approved by the Swiss Veterinary Health Authorities. The same protocol was used as previously described regarding anesthesia and analgesia with subcutaneous fentanyl-fluanisone (0.25 ml/kg) and medetomidini hydrochloridum (0.25 ml/kg). 19 
Aspiration Protocol
For the intratracheal injection of acid or phosphate-buffered saline (PBS) (control animals), the trachea was exposed surgically and a 22-gauge needle was inserted. Animals received 1 ml/kg of an endotoxin-free acidic solution (0.1 n, pH 1). Rats were exsanguinated at predefined time points (time after HCl instillation, 0.5, 1, 2, 4, 6, and 8 h; control animals, 4 h after PBS application). The vascular system was flushed with cold PBS. For bronchoalveolar lavage, 10 ml of cold PBS was gently instilled into the lungs, withdrawn, reinstilled repeatedly (four times), and collected. Cell pellets from centrifuged bronchoalveolar lavage fluid (BALF) were assessed for differential cell counts using cytospins and Diff-Quick (Dade Behring, Düdingen, Switzerland).
Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described previously. 20 Briefly, lungs or L2 cells were homogenized in ice cold buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm DTT, and 0.5 mm PMSF). Lysis was induced with 0.25% NP-40. After centrifugation, nuclear pellets were resuspended in ice cold buffer C (20 mm HEPES, pH 7.9, 0.4 M NaCl, 1 mm EDTA, 1 mm EGTA, 10% glycero1, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride) and rocked vigorously at 4°C for 1 h. The extracts were centrifuged and supernatants were frozen at −70°C. The protein concentration of the extracts was determined using the DC Protein Assay (Bio Rad, Hercules, CA). Nuclear extracts were then analyzed for the presence of NF-κB by electrophoretic mobility shift assay (EMSA), using a double-stranded oligonucleotide probe with the sequence 5′-AGT TGA G GG GAC TTT CCCAGG C-3′ (consensus site underlined) (Promega, Madison, WI). End labeling was accomplished by treatment with T4 kinase in the presence of [γ-32P]ATP. Extracts were then incubated for 50 min at room temperature with labeled NF-κB oligonucleotide in binding buffer in the presence or absence of unlabeled competitor oligonucleotide. Antibody supershift EMSA was performed by incubating nuclear extract proteins (10 μg) with 2 μg of a polyclonal rabbit antibody against p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). DNA–protein complexes were resolved in nondenaturing 5% acrylamide gels in 0.03% Tris boric acid plus EDTA acid buffer at 270 V. Gels were dried and exposed to X-OMAT KODAK film (Sigma) and developed after 6 h of exposure.
RNA Extraction and Reverse Transcriptase–Polymerase Chain Reaction Analysis
Total RNA from L2 cells and lungs was extracted as described previously. 15 Random hexanucleotide primers and murine leukemia virus reverse transcriptase were used for cDNA synthesis. Reverse transcriptase was performed with 0.8 μg RNA at 20°C for 5 min, 42°C for 30 min, and 99°C for 5 min. Specific primers located on separate exons were designed to assess gene expression of IκBα, ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2 in L2 cells and in total lung. For each primer pair, optimal cycling conditions were tested to detect target genes within the logarithmic amplification phase (table 1). Reverse transcriptase–polymerase chain reaction products were resolved on 1.5% agarose gels (Ultra Pure; Life Technologies) and stained with ethidium bromide. Gels were then photographed under ultraviolet light. Polymerase chain reaction was also performed with 18S primers to ensure equal loading.
Table 1. Optimized Conditions for Reverse Transcriptase–Polymerase Chain Reaction for mRNA
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Table 1. Optimized Conditions for Reverse Transcriptase–Polymerase Chain Reaction for mRNA
×
Lung Tissue Myeloperoxidase Content
Lungs were homogenized in a buffer containing 50 mm potassium phosphate and 0.5% hexadecyltrimethylammonium bromide and 5 mm EDTA, sonicated, and centrifuged as described earlier. 21 Fifty microliters of supernatant was added to 1,450 μl assay buffer, consisting of 100 mm potassium phosphate, o-dianisidine hydrochloride, and 30% H2O2. The reaction was assayed every 10 s at 420 nm (enzyme-linked immunosorbent assay reader). The results are shown as the slope of change in optical density over 360 s. Control values were defined as 1, whereas results from stimulated lungs were normalized to the value of 1.
Blocking Studies
Pyrrolidine dithiocarbamate, an inhibitor of NF-κB activation, was tested in the respiratory compartment. In a first set of experiments, the dose–response of different concentrations of PDTC was tested for optimal efficacy, whereby 50 μm was the most effective dosage (data not shown). Three hundred microliters of PDTC (50 μm) in PBS was instilled intratracheally, followed by intratracheal application of 300 μl HCl 45 min later. Endpoints of the investigation were neutrophil recruitment to the lung and expression of inflammatory mediators 5 h after HCl instillation. This time point represented the beginning of the second peak of the injury.
Statistical Analysis
All the results are expressed as means ± SEM. All experiments were conducted at least five times. ANOVA with post-ANOVA comparison was performed to assess the statistical significance of differences. Densitometry data are expressed as percentage values of controls (control, 100%). P  values < 0.05 were considered significant.
Results
Cytotoxicity Assays
To evaluate the cytotoxic effects of different pH values, 51Cr release was measured in the supernatant of stimulated L2 cells. 51Cr-labeled monolayers of L2 cells were exposed for 24 h to the following pH values: 7.4, 6, 5, 4, 3, and 2. Incubation of L2 cells with normal medium (pH, 7.4) was considered as control. As seen in figure 1A, pH values less than 6 lead to an increased accumulation of 51Cr in the supernatant. To further quantify cytotoxicity, L2 cells were incubated with medium with a pH value of 6 or 5 and supernatants were analyzed after 2, 4, and 6 h (figs. 1B and C). Although a pH value of 6 was not associated with cytotoxicity (fig. 1B), at a pH value of 5, cell death was observed (32 ± 2.1% cytotoxicity after 2 h, 36 ± 1.5% after 4 h, and 52 ± 7.0% after 6 h, all P  < 0.001, comparison between pH = 7.4 with pH = 5) fig. 1C).
Fig. 1. (A  ) Determination of cytotoxicity at different pH values. L2 cells were labeled with 51Cr, followed by a 24-h stimulation with pH values of 7.4, 6, 5, 4, 3, and 2. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks  indicate statistically significant difference from pH value induced cytotoxicity compared with pH of 7.4. *P  < 0.01. (B  ) Determination of cytotoxicity after stimulation of L2 cells with a pH value of 6. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 6 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Differences that are not significantly different from control stimulation (pH = 7.4) are indicated (n.s.). (C  ) Determination of cytotoxicity after stimulation of L2 with a pH value of 5. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 5 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks  indicate statistically significant difference from pH value of 5 induced cytotoxicity compared with pH of 7.4. *P  < 0.001.
Fig. 1. (A 
	) Determination of cytotoxicity at different pH values. L2 cells were labeled with 51Cr, followed by a 24-h stimulation with pH values of 7.4, 6, 5, 4, 3, and 2. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks 
	indicate statistically significant difference from pH value induced cytotoxicity compared with pH of 7.4. *P 
	< 0.01. (B 
	) Determination of cytotoxicity after stimulation of L2 cells with a pH value of 6. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 6 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Differences that are not significantly different from control stimulation (pH = 7.4) are indicated (n.s.). (C 
	) Determination of cytotoxicity after stimulation of L2 with a pH value of 5. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 5 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks 
	indicate statistically significant difference from pH value of 5 induced cytotoxicity compared with pH of 7.4. *P 
	< 0.001.
Fig. 1. (A  ) Determination of cytotoxicity at different pH values. L2 cells were labeled with 51Cr, followed by a 24-h stimulation with pH values of 7.4, 6, 5, 4, 3, and 2. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks  indicate statistically significant difference from pH value induced cytotoxicity compared with pH of 7.4. *P  < 0.01. (B  ) Determination of cytotoxicity after stimulation of L2 cells with a pH value of 6. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 6 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Differences that are not significantly different from control stimulation (pH = 7.4) are indicated (n.s.). (C  ) Determination of cytotoxicity after stimulation of L2 with a pH value of 5. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 5 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks  indicate statistically significant difference from pH value of 5 induced cytotoxicity compared with pH of 7.4. *P  < 0.001.
×
NF-κB Activation in L2 Cells after Stimulation with HCl
Based on the aforementioned experiments, we stimulated L2 cells with medium at pH 6 for various time points (0.5, 1, 2, 4, 6, 8, and 24 h). NF-κB was extracted after stimulation with HCl, and NF-κB binding activity of HCl-stimulated cells was evaluated with EMSA. As shown in figure 2, a clear biphasic activation pattern of NF-κB activity was seen. NF-κB binding activity was increased at 0.5 h by 82 ± 23% (not significant), at 1 h by 161 ± 16% (P  < 0.01), at 2 h by 0 ± 9% (not significant), at 4 h by 125 ± 2% (P  < 0.001), at 6 h by 179 ± 7% (P  < 0.001), at 8 h by 211 ± 25% (P  < 0.001), and at 24 h by 118 ± 9% (P  < 0.005) compared with NF-κB binding activity from control cells.
Fig. 2. Determination of NF-κB DNA binding activity. Nuclear elements were extracted from L2 cells, previously exposed to a pH value of 6 for 0.5, 1, 2, 4, 6, 8, and 24 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bars  represent values from control cells, and black bars  represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 2. Determination of NF-κB DNA binding activity. Nuclear elements were extracted from L2 cells, previously exposed to a pH value of 6 for 0.5, 1, 2, 4, 6, 8, and 24 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bars 
	represent values from control cells, and black bars 
	represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 2. Determination of NF-κB DNA binding activity. Nuclear elements were extracted from L2 cells, previously exposed to a pH value of 6 for 0.5, 1, 2, 4, 6, 8, and 24 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bars  represent values from control cells, and black bars  represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
×
Effects of HCl on mRNA for ICAM-1, TNF-α, MCP-1, and MIP-1β in L2 Cells
The time-dependent effect of the stimulation with an acidic environment at a pH value of 6 was evaluated on mRNA expression of several inflammatory mediators. L2 cells were thereby stimulated for 0.5, 1, 2, 4, 6, 8, and 24 h. TNF-α was not detectable in L2 cells (also verified with lipopolysaccharide stimulation as performed in previous studies 22), but mRNA for ICAM-1 was progressively up-regulated in L2 cells from 2 to 8 h between 254 ± 3% at 2 h (P  < 0.001) and 258 ± 18% at 8 h (P  < 0.001) (fig. 3). MCP-1 showed a similar expression pattern with a 434 ± 34% increase after 0.5 h (P  < 0.005) and a sustained enhanced expression for 1, 2, 4, 6, 8, and 24 h (all P  < 0.005). mRNA for MIP-1β, however, appeared to be up-regulated between 0.5 and 1 h (208 ± 39%, 373 ± 58%, P  < 0.05) and between 4 h and 8 h (193 ± 31%, 335 ± 27%, 212 ± 4%) (P  < 0.05, P  < 0.01, and P  < 0.001, respectively).
Fig. 3. Determination of mRNA of ICAM-1, MCP-1, and MIP-1β in L2 cells (TNF-α was not detectable). Cells were exposed to pH value of 6 for various periods. RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bar  represents values from control cells, and black bars  represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments.
Fig. 3. Determination of mRNA of ICAM-1, MCP-1, and MIP-1β in L2 cells (TNF-α was not detectable). Cells were exposed to pH value of 6 for various periods. RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bar 
	represents values from control cells, and black bars 
	represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot 
	represents one of five experiments.
Fig. 3. Determination of mRNA of ICAM-1, MCP-1, and MIP-1β in L2 cells (TNF-α was not detectable). Cells were exposed to pH value of 6 for various periods. RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bar  represents values from control cells, and black bars  represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments.
×
Activation of Lung NF-κB during Acid-induced Lung Inflammation
The kinetics of NF-κB binding activity during acid-induced lung inflammation were determined by EMSA of nuclear extracts from whole lung obtained at various time points (0.5, 1, 2, 4, 6, and 8 h) after instillation of acidic solution (pH 1). As shown in figure 4A, low levels of constitutive NF-κB binding activity were found in lung extracts without HCl stimulation. NF-κB binding activity was increased within 0.5 h to 689 ± 135% (P  < 0.05) to 1 h and 2 h with 608 ± 93% (P  < 0.05) and 500 ± 15% (P  < 0.001) after airway instillation of HCl and decreased to baseline activity thereafter. A second maximum was reached at 8 h with 697 ± 35% (P  < 0.005), reflecting a biphasic activation pattern during the observed time. Controls were run with cold oligonucleotides and p65 antibody. To further explore the expression characteristics at the level of transcription factors, IκBα expression pattern was investigated. A first maximum was detected at 1 h (581 ± 66% increase) and at 2 h (596 ± 81% increase) (both P  < 0.01), with a second maximum at 8 h with 674 ± 12% of up-regulation (P  < 0.001) (fig. 4B), reflecting the findings with NF-κB.
Fig. 4. (A  ) Determination of NF-κB DNA binding activity. Nuclear elements were extracted from whole lung, previously exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bar  represents values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments. (B  ) Determination of whole lung mRNA of IκBα. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 4. (A 
	) Determination of NF-κB DNA binding activity. Nuclear elements were extracted from whole lung, previously exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bar 
	represents values from control animals, and black bars 
	represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot 
	represents one of five experiments. (B 
	) Determination of whole lung mRNA of IκBα. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 4. (A  ) Determination of NF-κB DNA binding activity. Nuclear elements were extracted from whole lung, previously exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bar  represents values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments. (B  ) Determination of whole lung mRNA of IκBα. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
×
Enhanced Expression of Whole Lung mRNA for ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2
mRNA was obtained from whole lung tissue at 0.5, 1, 2, 4, 6, and 8 h after instillation of HCl. mRNA content for ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2 was evaluated with reverse transcriptase–polymerase chain reaction analysis and densitometry of the data (fig. 5). ICAM-1 mRNA showed a baseline expression in control animals receiving PBS. An increase in mRNA expression was observed between 1 and 8 h. For TNF-α, mRNA was enhanced after 0.5 h of HCl stimulation and remained up-regulated up to 8 h. mRNA for MCP-1 showed a maximum between 0.5 and 8 h. It is noteworthy that the chemokines MIP-1β and MIP-2 presented a biphasic expression pattern with up-regulation maxima similar to those seen with NF-κB; an early increase after 0.5 h (283 ± 30%, P  < 0.01 and 369 ± 6%, respectively, P  < 0.001) to 1 h (353 ± 77% and 267 ± 136%, respectively, both P  < 0.05). A second peak was seen at 8 h (421 ± 9%, P  < 0.001, and 274 ± 41%, P  < 0.05).
Fig. 5. Determination of whole lung mRNA of ICAM-1, TNF-1α, MCP-1, MIP-1β, and MIP-2. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from phosphate-buffered saline animals (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments.
Fig. 5. Determination of whole lung mRNA of ICAM-1, TNF-1α, MCP-1, MIP-1β, and MIP-2. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from phosphate-buffered saline animals (100%). Values are means ± SEM from five different assays. The blot 
	represents one of five experiments.
Fig. 5. Determination of whole lung mRNA of ICAM-1, TNF-1α, MCP-1, MIP-1β, and MIP-2. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from phosphate-buffered saline animals (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments.
×
Time Course of Neutrophil Accumulation
Recruitment of neutrophils to interstitial and alveolar space in response to acid instillation was documented by a myeloperoxidase assay and neutrophil counts in BALF. For quantification of interstitial neutrophil accumulation, lungs were homogenized at time points from 0.5 to 8 h after HCl application, and a myeloperoxidase assay was performed. A first maximum increase of myeloperoxidase was observed at 0.5 and 1 h (554 ± 0.1%, P  < 0.01, and 673 ± 0.5% enhancement, P  < 0.05, respectively), with a second maximum at 8 h with a 536 ± 0.01% increase (P  < 0.001) (fig. 6A). For quantification of alveolar neutrophil accumulation, cells were counted in the BALF in control animals (PBS for 4 h) and at time points from 0.5 to 8 h after HCl instillation. As shown in figure 6B, total cell content increased from 0.99 × 106± 0.1 × 106cells/ml in lungs after PBS instillation to a peak of 7.33 ×106± 1.2 ×106cells/ml at 8 h after HCl exposure (600% increase, P  < 0.001). Whereas with PBS animals alveolar macrophages were the only cell type in the lavage, neutrophil accumulation increased after HCl instillation. At 0.5 h, 32 ± 2.1% of the cells were neutrophils, at 1 h, 50 ± 2.5% (P  < 0.05); at 2 h, 24 ± 2.0% (P  < 0.01); at 4 h, 51 ± 12.0%P  < 0.05); at 6 h, 50 ± 2.5% (P  < 0.001); and at 8 h, 69 ± 3.8% (P  < 0.001) (fig. 6C). Again, a biphasic pattern was observed with neutrophil accumulation in the respiratory compartment (0.5 and 1 h, 6 and 8 h).
Fig. 6. (A  ) Neutrophil accumulation in the lung. Lungs of acid-exposed and control animals were homogenized, and myeloperoxidase was analyzed. Values are means ± SEM from five animals. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Asterisks  indicate maximally enhanced myeloperoxidase values of acid-exposed animals with statistically significant difference compared to control animals. *P  < 0.01. (B  ) Cell analysis in bronchoalveolar lavage fluid (BALF). Rats were exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Cells were analyzed by Diff-Quick. The gray portions  of the bars  represent alveolar macrophages, whereas the black portions  reflect neutrophils. Values are means ± SEM from five animals. Asterisks  indicate statistically significant difference from animals exposed to acid compared with control animals. *P  < 0.05. (C  ) Cell analysis in bronchoalveolar lavage fluid (BALF) showing percentage of neutrophils. Values are means ± SEM from five animals. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Asterisks  indicate statistically significant difference concerning neutrophil accumulation from animals exposed to acid compared with control animals. *P  < 0.05.
Fig. 6. (A 
	) Neutrophil accumulation in the lung. Lungs of acid-exposed and control animals were homogenized, and myeloperoxidase was analyzed. Values are means ± SEM from five animals. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Asterisks 
	indicate maximally enhanced myeloperoxidase values of acid-exposed animals with statistically significant difference compared to control animals. *P 
	< 0.01. (B 
	) Cell analysis in bronchoalveolar lavage fluid (BALF). Rats were exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Cells were analyzed by Diff-Quick. The gray portions 
	of the bars 
	represent alveolar macrophages, whereas the black portions 
	reflect neutrophils. Values are means ± SEM from five animals. Asterisks 
	indicate statistically significant difference from animals exposed to acid compared with control animals. *P 
	< 0.05. (C 
	) Cell analysis in bronchoalveolar lavage fluid (BALF) showing percentage of neutrophils. Values are means ± SEM from five animals. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Asterisks 
	indicate statistically significant difference concerning neutrophil accumulation from animals exposed to acid compared with control animals. *P 
	< 0.05.
Fig. 6. (A  ) Neutrophil accumulation in the lung. Lungs of acid-exposed and control animals were homogenized, and myeloperoxidase was analyzed. Values are means ± SEM from five animals. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Asterisks  indicate maximally enhanced myeloperoxidase values of acid-exposed animals with statistically significant difference compared to control animals. *P  < 0.01. (B  ) Cell analysis in bronchoalveolar lavage fluid (BALF). Rats were exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Cells were analyzed by Diff-Quick. The gray portions  of the bars  represent alveolar macrophages, whereas the black portions  reflect neutrophils. Values are means ± SEM from five animals. Asterisks  indicate statistically significant difference from animals exposed to acid compared with control animals. *P  < 0.05. (C  ) Cell analysis in bronchoalveolar lavage fluid (BALF) showing percentage of neutrophils. Values are means ± SEM from five animals. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Asterisks  indicate statistically significant difference concerning neutrophil accumulation from animals exposed to acid compared with control animals. *P  < 0.05.
×
Blocking Effect of PDTC in Vivo 
PDTC, an inhibitor of NF-κB activation, has been shown to possess antiinflammatory activity. The possible antiinflammatory effect of intratracheally applied PDTC (50 μm, 300 μl) was evaluated in vivo  in the acid aspiration lung model. Neutrophil content in the interstitial space decreased by 65% (P  < 0.01) (fig. 7A). Furthermore, BALF in control animals (PBS 1 h, HCl 5 h) contained 10 × 106± 0.6 × 106cells, whereas treated animals (PDTC 1 h, HCl 5 h) contained only 6.2 × 106± 0.2 × 106cells (55% reduction, P  < 0.01) (fig. 7B). On the level of adhesion molecules, no difference between PBS or PDTC animals in ICAM-1 expression after HCl exposure was observed. However, expression of mRNA for TNF-α, MCP-1, MIP-1β, and MIP-2 were decreased under PDTC (fig. 7C). TNF-α mRNA was decreased by 58 ± 7.5% (P  < 0.05), MCP-1 by 59 ± 1% (P  < 0.005), MIP-1β by 54 ± 12% (P  < 0.05), and MIP-2 by 57 ± 7% (P  < 0.05).
Fig. 7. (A  ) Neutrophil accumulation in the lung after intratracheal blocking with pyrrolidine dithiocarbamate (PDTC). PDTC or phosphate-buffered saline (PBS) as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were homogenized, and myeloperoxidase was determined. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Values are means ± SEM from five animals. Asterisk indicates statistically significant difference from animals exposed to PDTC and acid compared with PBS and acid. *P  < 0.01. (B  ) Cell analysis in bronchoalveolar lavage fluid (BALF) after intratracheal blocking with PDTC. PDTC or PBS as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were lavaged, and cells were analyzed by Diff-Quick. The gray part of the bars represents alveolar macrophages, whereas the black part reflects neutrophils. Asterisks  indicate statistically significant difference concerning neutrophil accumulation from animals exposed to PDTC and acid compared with PBS and acid. Values are means ± SEM from five animals. *P  < 0.01. (C  ) Determination of whole lung mRNA of ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2. Animals were pretreated with PBS or PDTC, followed by HCl application for 5 h. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction (RT-PCR) was performed. Equal loading was shown with 18S bands. Densitometry was performed, and results are expressed as the percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 7. (A 
	) Neutrophil accumulation in the lung after intratracheal blocking with pyrrolidine dithiocarbamate (PDTC). PDTC or phosphate-buffered saline (PBS) as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were homogenized, and myeloperoxidase was determined. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Values are means ± SEM from five animals. Asterisk indicates statistically significant difference from animals exposed to PDTC and acid compared with PBS and acid. *P 
	< 0.01. (B 
	) Cell analysis in bronchoalveolar lavage fluid (BALF) after intratracheal blocking with PDTC. PDTC or PBS as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were lavaged, and cells were analyzed by Diff-Quick. The gray part of the bars represents alveolar macrophages, whereas the black part reflects neutrophils. Asterisks 
	indicate statistically significant difference concerning neutrophil accumulation from animals exposed to PDTC and acid compared with PBS and acid. Values are means ± SEM from five animals. *P 
	< 0.01. (C 
	) Determination of whole lung mRNA of ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2. Animals were pretreated with PBS or PDTC, followed by HCl application for 5 h. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction (RT-PCR) was performed. Equal loading was shown with 18S bands. Densitometry was performed, and results are expressed as the percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 7. (A  ) Neutrophil accumulation in the lung after intratracheal blocking with pyrrolidine dithiocarbamate (PDTC). PDTC or phosphate-buffered saline (PBS) as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were homogenized, and myeloperoxidase was determined. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Values are means ± SEM from five animals. Asterisk indicates statistically significant difference from animals exposed to PDTC and acid compared with PBS and acid. *P  < 0.01. (B  ) Cell analysis in bronchoalveolar lavage fluid (BALF) after intratracheal blocking with PDTC. PDTC or PBS as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were lavaged, and cells were analyzed by Diff-Quick. The gray part of the bars represents alveolar macrophages, whereas the black part reflects neutrophils. Asterisks  indicate statistically significant difference concerning neutrophil accumulation from animals exposed to PDTC and acid compared with PBS and acid. Values are means ± SEM from five animals. *P  < 0.01. (C  ) Determination of whole lung mRNA of ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2. Animals were pretreated with PBS or PDTC, followed by HCl application for 5 h. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction (RT-PCR) was performed. Equal loading was shown with 18S bands. Densitometry was performed, and results are expressed as the percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
×
Discussion
The goal of this study was to analyze the role of transcription factor NF-κB and its inhibitor PDTC in a model of acid-induced lung inflammation. NF-κB is an inflammatory mediator, and its expression on stimulation is enhanced at the beginning of an inflammatory cascade. It is a promising target for a potential blocking substance because all ensuing steps of the cascade are also stopped.
Alveolar epithelial cells are the main target cells in the epithelial respiratory compartment exposed to noxious substances such as lipopolysaccharide or acid. Therefore, we examined the effect of acid on these cells in vitro  . It is noteworthy that a pH value less than 6 induced cytotoxicity, as shown with chromium assays. Acid at higher pH values, however, did not cause any toxic effect, but induced an inflammatory reaction in the cell. This is an interesting finding that would lead to the assumption that aspiration of stomach contents with a low pH in a first step causes death of AEC, which by itself could induce an inflammatory response through the recruitment of effector cells in vivo  .
It is known that acid aspiration has a biphasic pathogenesis. 3 A first injury peak was described at 1 h, with a second one after 4 h. Our studies support these findings, but the biphasic character at the level of transcriptional factors has never been shown in acid aspiration. A biphasic activation pattern of NF-κB to different stimuli has been confirmed in other experimental models. In vitro  hepatocyte stimulation by TNF-α has been described to cause an early induction of NF-κB binding activity (15–30 min), followed by a decrease to control levels after 60 min and then a late induction after 120 min. 23 Ischemia and reperfusion in rat skeletal muscle in vivo  also resulted in a biphasic activation pattern of NF-κB binding activity, with peak activities observed from 0.5 to 3 h after perfusion and from 6 to 16 h after perfusion and a return to baseline activity between 3 and 6 h. 24 In vitro  experiments with human epithelial cells have shown that acidic exposure increased the activation of the transcription factor NF-κB. 25,26 However, there was no biphasic expression pattern detectable. Xu et al.  25 worked with human ovarian cancer cells that could have a different character of responding to an injury than AEC. In addition, cancer cells may not show the same expression pattern of NF-κB binding activity as healthy ovarian cells. In the study by Ishizuka et al.  , 26 the focus on airway epithelial cells was limited to 60 min, which does not exclude a second peak of NF-κB binding activity at a later time point.
Control over NF-κB binding activity in response to a given stimulus is maintained through intracellular and extracellular feedback control mechanisms. 27,28 Intracellularly, the activation of NF-κB binding activity is prevented by inhibitory cytoplasmic proteins of the IκB family, consisting of different subunits, from which the best studied are IκB-α and IκB-β. 29 The major difference between IκB-α and IκB-β lies in the response to different inducers of NF-κB activity. Whereas the IκB-α response, which is defined as degradation of the inhibitory protein IκB-α, is used for responding immediately to transient situations of stimulation, 30 the degradation of IkB-β may be used for persistent response in inflammation, infection, or differentiation. 31,32 The up-regulated IκB-α protein helps to shut down the NF-κB response, thus ensuring that genes are activated only transiently. A biphasic activity pattern of NF-κB could be explained as follows: The early peak may be the result of degradation of IκB-α complexes and subsequent inhibition of the NF-κB activity by up-regulated IκB-α protein via  the described autoregulatory feedback mechanism. The later peak of NF-κB activity, however, could be the consequence of degradation of IκB-β complexes. 33 In addition to intracellular feedback control, extracellular control mechanisms for the reduction of NF-κB activation may also be in place. In vivo  studies have shown that interleukin-10 and interleukin-13, two counterregulatory cytokines produced by inflammatory stimuli, inhibit NF-κB activation in association with preserved expression of IκB-α. 28 Further investigations must explore the precise mechanisms that control intracellular and extracellular feedback in NF-κB activity in the acid-induced lung injury.
The chemokines MIP-1β and MIP-2 show a similar biphasic expression pattern, as does NF-κB. Therefore, it appears likely that neutrophil recruitment into the lung at these two different time points is probably triggered by NF-κB via  MIP-1β and MIP-2. It is noteworthy that the expression of the NF-κB target genes ICAM-1, TNF-α, and MCP-1 examined in this study did not reflect the biphasic activating pattern of NF-κB binding activity. A missing two-peak expression pattern of ICAM-1, TNF-α, and MCP-1 could be the result of the influence of other regulatory factors on the transcription of these genes. NF-κB is not a unique transcription factor, and a gene may be regulated by other transcription factors so that an interrelated system of regulatory mechanisms finally ends up in the transcription of a single gene. A second explanation would be that the activation pattern of the transcription factor may not be reflected in the concentration of the cellular mRNA at the same time points, because stability of different mRNAs in the cytoplasm varies widely. 34 Further experiments must be performed to study more inflammatory mediators to identify potential chemoattractants for neutrophils that reflect biphasic expression patterns.
Comparing in vitro  and in vivo  expression patterns of inflammatory mediators shows only moderate differences regarding mRNA for ICAM-1, MCP-1, and MIP-1β. This similarity may exist because AECs are the target cells in vivo  during acid-induced injury. The difference in TNF-α expression in vitro  and in vivo  probably results from the character of L2 cells, which are only a cell line and not a primary culture of AECs. Therefore, the cell line may not have the true character of AECs.
Although PDTC is not an exclusive NF-κB inhibitor, its blocking effect on NF-κB was tested. We postulated that administration of PDTC could affect the acid-induced inflammatory response as shown in other models. 35 Because the lungs are easily accessible via  the respiratory epithelial compartment, a potential effect was evaluated through intratracheal application. This approach would allow the potential effect to be more confined to the lungs, with a possible reduction of undesired systemic effects. After application of PDTC, induction of ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2 was studied, as was neutrophil accumulation in the interstitial space and in the BALF. It is noteworthy that whole lung mRNA for ICAM-1 was not affected by PDTC. Although this gene is NF-κB-dependent, a lack of an effect of PDTC on ICAM-1 could also be shown on human endothelial cells. 36 However, recruitment of neutrophils was significantly reduced by PDTC, a successful step toward a possible therapeutic effect. Neutrophils are important effector cells that do cause injury to alveolar-capillary unit through nonoxidative and oxidative mechanisms. 4 Attenuated mRNA levels of TNF-α, MCP-1, MIP-1β, and MIP-2 could explain the mechanism behind diminished neutrophil accumulation.
In summary, these data show an activation pattern of transcription factor NF-κB characterized by two phases after acid-induced injury in vitro  in AECs and in vivo  in rat lung. Blocking studies with intratracheally applied PDTC attenuated lung injury by significantly decreasing neutrophil recruitment. It will be important to dissect the different stages of the NF-κB activation in this animal model of acid-induced lung injury, which may lead to specific modification of the inflammatory response by therapeutic interventions.
The authors thank Christian Gasser, art worker, Institute of Physiology, University of Zurich, for the development of illustrations.
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Fig. 1. (A  ) Determination of cytotoxicity at different pH values. L2 cells were labeled with 51Cr, followed by a 24-h stimulation with pH values of 7.4, 6, 5, 4, 3, and 2. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks  indicate statistically significant difference from pH value induced cytotoxicity compared with pH of 7.4. *P  < 0.01. (B  ) Determination of cytotoxicity after stimulation of L2 cells with a pH value of 6. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 6 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Differences that are not significantly different from control stimulation (pH = 7.4) are indicated (n.s.). (C  ) Determination of cytotoxicity after stimulation of L2 with a pH value of 5. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 5 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks  indicate statistically significant difference from pH value of 5 induced cytotoxicity compared with pH of 7.4. *P  < 0.001.
Fig. 1. (A 
	) Determination of cytotoxicity at different pH values. L2 cells were labeled with 51Cr, followed by a 24-h stimulation with pH values of 7.4, 6, 5, 4, 3, and 2. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks 
	indicate statistically significant difference from pH value induced cytotoxicity compared with pH of 7.4. *P 
	< 0.01. (B 
	) Determination of cytotoxicity after stimulation of L2 cells with a pH value of 6. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 6 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Differences that are not significantly different from control stimulation (pH = 7.4) are indicated (n.s.). (C 
	) Determination of cytotoxicity after stimulation of L2 with a pH value of 5. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 5 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks 
	indicate statistically significant difference from pH value of 5 induced cytotoxicity compared with pH of 7.4. *P 
	< 0.001.
Fig. 1. (A  ) Determination of cytotoxicity at different pH values. L2 cells were labeled with 51Cr, followed by a 24-h stimulation with pH values of 7.4, 6, 5, 4, 3, and 2. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks  indicate statistically significant difference from pH value induced cytotoxicity compared with pH of 7.4. *P  < 0.01. (B  ) Determination of cytotoxicity after stimulation of L2 cells with a pH value of 6. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 6 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Differences that are not significantly different from control stimulation (pH = 7.4) are indicated (n.s.). (C  ) Determination of cytotoxicity after stimulation of L2 with a pH value of 5. L2 cells were labeled with 51Cr, followed by a stimulation with a pH value of 5 for 2 h, 4 h, and 6 h. 51Cr in the supernatant was determined, and cytotoxicity was calculated. Values are means ± SEM from five different assays. Asterisks  indicate statistically significant difference from pH value of 5 induced cytotoxicity compared with pH of 7.4. *P  < 0.001.
×
Fig. 2. Determination of NF-κB DNA binding activity. Nuclear elements were extracted from L2 cells, previously exposed to a pH value of 6 for 0.5, 1, 2, 4, 6, 8, and 24 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bars  represent values from control cells, and black bars  represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 2. Determination of NF-κB DNA binding activity. Nuclear elements were extracted from L2 cells, previously exposed to a pH value of 6 for 0.5, 1, 2, 4, 6, 8, and 24 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bars 
	represent values from control cells, and black bars 
	represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 2. Determination of NF-κB DNA binding activity. Nuclear elements were extracted from L2 cells, previously exposed to a pH value of 6 for 0.5, 1, 2, 4, 6, 8, and 24 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bars  represent values from control cells, and black bars  represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
×
Fig. 3. Determination of mRNA of ICAM-1, MCP-1, and MIP-1β in L2 cells (TNF-α was not detectable). Cells were exposed to pH value of 6 for various periods. RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bar  represents values from control cells, and black bars  represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments.
Fig. 3. Determination of mRNA of ICAM-1, MCP-1, and MIP-1β in L2 cells (TNF-α was not detectable). Cells were exposed to pH value of 6 for various periods. RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bar 
	represents values from control cells, and black bars 
	represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot 
	represents one of five experiments.
Fig. 3. Determination of mRNA of ICAM-1, MCP-1, and MIP-1β in L2 cells (TNF-α was not detectable). Cells were exposed to pH value of 6 for various periods. RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bar  represents values from control cells, and black bars  represent values after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from control cells (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments.
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Fig. 4. (A  ) Determination of NF-κB DNA binding activity. Nuclear elements were extracted from whole lung, previously exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bar  represents values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments. (B  ) Determination of whole lung mRNA of IκBα. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 4. (A 
	) Determination of NF-κB DNA binding activity. Nuclear elements were extracted from whole lung, previously exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bar 
	represents values from control animals, and black bars 
	represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot 
	represents one of five experiments. (B 
	) Determination of whole lung mRNA of IκBα. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 4. (A  ) Determination of NF-κB DNA binding activity. Nuclear elements were extracted from whole lung, previously exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Electrophoretic mobility shift assay for NF-κB was performed. Specificity for NF-κB was shown by using competitive unlabeled NF-κB oligonucleotides and a p65 antibody. White bar  represents values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments. (B  ) Determination of whole lung mRNA of IκBα. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
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Fig. 5. Determination of whole lung mRNA of ICAM-1, TNF-1α, MCP-1, MIP-1β, and MIP-2. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from phosphate-buffered saline animals (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments.
Fig. 5. Determination of whole lung mRNA of ICAM-1, TNF-1α, MCP-1, MIP-1β, and MIP-2. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from phosphate-buffered saline animals (100%). Values are means ± SEM from five different assays. The blot 
	represents one of five experiments.
Fig. 5. Determination of whole lung mRNA of ICAM-1, TNF-1α, MCP-1, MIP-1β, and MIP-2. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction was performed. Equal loading was shown with 18S bands. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Densitometry was performed, and results are expressed as percentage of values obtained from phosphate-buffered saline animals (100%). Values are means ± SEM from five different assays. The blot  represents one of five experiments.
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Fig. 6. (A  ) Neutrophil accumulation in the lung. Lungs of acid-exposed and control animals were homogenized, and myeloperoxidase was analyzed. Values are means ± SEM from five animals. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Asterisks  indicate maximally enhanced myeloperoxidase values of acid-exposed animals with statistically significant difference compared to control animals. *P  < 0.01. (B  ) Cell analysis in bronchoalveolar lavage fluid (BALF). Rats were exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Cells were analyzed by Diff-Quick. The gray portions  of the bars  represent alveolar macrophages, whereas the black portions  reflect neutrophils. Values are means ± SEM from five animals. Asterisks  indicate statistically significant difference from animals exposed to acid compared with control animals. *P  < 0.05. (C  ) Cell analysis in bronchoalveolar lavage fluid (BALF) showing percentage of neutrophils. Values are means ± SEM from five animals. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Asterisks  indicate statistically significant difference concerning neutrophil accumulation from animals exposed to acid compared with control animals. *P  < 0.05.
Fig. 6. (A 
	) Neutrophil accumulation in the lung. Lungs of acid-exposed and control animals were homogenized, and myeloperoxidase was analyzed. Values are means ± SEM from five animals. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Asterisks 
	indicate maximally enhanced myeloperoxidase values of acid-exposed animals with statistically significant difference compared to control animals. *P 
	< 0.01. (B 
	) Cell analysis in bronchoalveolar lavage fluid (BALF). Rats were exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Cells were analyzed by Diff-Quick. The gray portions 
	of the bars 
	represent alveolar macrophages, whereas the black portions 
	reflect neutrophils. Values are means ± SEM from five animals. Asterisks 
	indicate statistically significant difference from animals exposed to acid compared with control animals. *P 
	< 0.05. (C 
	) Cell analysis in bronchoalveolar lavage fluid (BALF) showing percentage of neutrophils. Values are means ± SEM from five animals. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Asterisks 
	indicate statistically significant difference concerning neutrophil accumulation from animals exposed to acid compared with control animals. *P 
	< 0.05.
Fig. 6. (A  ) Neutrophil accumulation in the lung. Lungs of acid-exposed and control animals were homogenized, and myeloperoxidase was analyzed. Values are means ± SEM from five animals. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Asterisks  indicate maximally enhanced myeloperoxidase values of acid-exposed animals with statistically significant difference compared to control animals. *P  < 0.01. (B  ) Cell analysis in bronchoalveolar lavage fluid (BALF). Rats were exposed to acid for 0.5, 1, 2, 4, 6, and 8 h. Cells were analyzed by Diff-Quick. The gray portions  of the bars  represent alveolar macrophages, whereas the black portions  reflect neutrophils. Values are means ± SEM from five animals. Asterisks  indicate statistically significant difference from animals exposed to acid compared with control animals. *P  < 0.05. (C  ) Cell analysis in bronchoalveolar lavage fluid (BALF) showing percentage of neutrophils. Values are means ± SEM from five animals. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Asterisks  indicate statistically significant difference concerning neutrophil accumulation from animals exposed to acid compared with control animals. *P  < 0.05.
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Fig. 7. (A  ) Neutrophil accumulation in the lung after intratracheal blocking with pyrrolidine dithiocarbamate (PDTC). PDTC or phosphate-buffered saline (PBS) as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were homogenized, and myeloperoxidase was determined. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Values are means ± SEM from five animals. Asterisk indicates statistically significant difference from animals exposed to PDTC and acid compared with PBS and acid. *P  < 0.01. (B  ) Cell analysis in bronchoalveolar lavage fluid (BALF) after intratracheal blocking with PDTC. PDTC or PBS as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were lavaged, and cells were analyzed by Diff-Quick. The gray part of the bars represents alveolar macrophages, whereas the black part reflects neutrophils. Asterisks  indicate statistically significant difference concerning neutrophil accumulation from animals exposed to PDTC and acid compared with PBS and acid. Values are means ± SEM from five animals. *P  < 0.01. (C  ) Determination of whole lung mRNA of ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2. Animals were pretreated with PBS or PDTC, followed by HCl application for 5 h. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction (RT-PCR) was performed. Equal loading was shown with 18S bands. Densitometry was performed, and results are expressed as the percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 7. (A 
	) Neutrophil accumulation in the lung after intratracheal blocking with pyrrolidine dithiocarbamate (PDTC). PDTC or phosphate-buffered saline (PBS) as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were homogenized, and myeloperoxidase was determined. White bars 
	represent values from control animals, and black bars 
	represent values from animals after HCl stimulation. Values are means ± SEM from five animals. Asterisk indicates statistically significant difference from animals exposed to PDTC and acid compared with PBS and acid. *P 
	< 0.01. (B 
	) Cell analysis in bronchoalveolar lavage fluid (BALF) after intratracheal blocking with PDTC. PDTC or PBS as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were lavaged, and cells were analyzed by Diff-Quick. The gray part of the bars represents alveolar macrophages, whereas the black part reflects neutrophils. Asterisks 
	indicate statistically significant difference concerning neutrophil accumulation from animals exposed to PDTC and acid compared with PBS and acid. Values are means ± SEM from five animals. *P 
	< 0.01. (C 
	) Determination of whole lung mRNA of ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2. Animals were pretreated with PBS or PDTC, followed by HCl application for 5 h. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction (RT-PCR) was performed. Equal loading was shown with 18S bands. Densitometry was performed, and results are expressed as the percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
Fig. 7. (A  ) Neutrophil accumulation in the lung after intratracheal blocking with pyrrolidine dithiocarbamate (PDTC). PDTC or phosphate-buffered saline (PBS) as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were homogenized, and myeloperoxidase was determined. White bars  represent values from control animals, and black bars  represent values from animals after HCl stimulation. Values are means ± SEM from five animals. Asterisk indicates statistically significant difference from animals exposed to PDTC and acid compared with PBS and acid. *P  < 0.01. (B  ) Cell analysis in bronchoalveolar lavage fluid (BALF) after intratracheal blocking with PDTC. PDTC or PBS as a control was instilled intratracheally, followed by HCl application or PBS as a control 45 min later for 5 h. Lungs were lavaged, and cells were analyzed by Diff-Quick. The gray part of the bars represents alveolar macrophages, whereas the black part reflects neutrophils. Asterisks  indicate statistically significant difference concerning neutrophil accumulation from animals exposed to PDTC and acid compared with PBS and acid. Values are means ± SEM from five animals. *P  < 0.01. (C  ) Determination of whole lung mRNA of ICAM-1, TNF-α, MCP-1, MIP-1β, and MIP-2. Animals were pretreated with PBS or PDTC, followed by HCl application for 5 h. Whole lung RNA was extracted, and reverse transcriptase–polymerase chain reaction (RT-PCR) was performed. Equal loading was shown with 18S bands. Densitometry was performed, and results are expressed as the percentage of values obtained from PBS animals (100%). Values are means ± SEM from five different assays. The blot represents one of five experiments.
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Table 1. Optimized Conditions for Reverse Transcriptase–Polymerase Chain Reaction for mRNA
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Table 1. Optimized Conditions for Reverse Transcriptase–Polymerase Chain Reaction for mRNA
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