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ABSTRACT |
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INTRODUCTION
METHODS
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Aspiration of gastric contents is one of leading causes of the acute
respiratory distress syndrome (ARDS). The pathogenesis of acid
aspiration-induced acute lung injury is well understood. Less clear
is why patients who have suffered acid aspiration are susceptible
to ARDS. We studied the effects of acid instillation on the inflammatory response to subsequent lipopolysaccharide (LPS) challenge in rats. Instillation of acid into the right lung worsened the
pathology induced by LPS that was administered 24 h after acid
instillation. This included worsened oxygenation, increased pulmonary edema, increased production of tumor necrosis factor-alpha (TNF-
) and cytokine-induced neutrophil chemoattractant, neutrophil accumulation and mobilization to the alveolar spaces, and nitric oxide (NO) production. Of interest, neutrophil mobilization, NO production, and protein permeability were also magnified in the left lung. These effects were attenuated by administration of the protein tyrosine kinase (PTK) inhibitors genistein and tyrphostin AG556. These data suggest that acid instillation primes the rat to enhance the inflammatory response to subsequent endotoxin challenge and that at least part of the augmented inflammatory response depends on PTK.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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More than three decades have passed since Ashbaugh and coworkers (1) first described acute respiratory distress syndrome (ARDS) in which increased protein permeability across the endothelial and epithelial barriers of the lung impedes oxygenation. Despite advances in supportive care, the mortality rate of patients with ARDS remains high, frequently in excess of 50% (2).
Aspiration of gastric contents is the second or third most common cause of ARDS (3). The pathogenesis of acid aspiration pneumonia itself is reasonably well understood. Whereas direct injury caused by the acid is limited, subsequent neutrophil activation triggers further lung injury and systemic effects (4). There is, however, no clear explanation of why patients who have suffered from acid aspiration are more susceptible to ARDS.
Acid aspiration frequently accompanies bacterial pneumonia, which increases the mortality. Microorganisms that predominate in aspiration pneumonia reflect the oropharyngeal normal flora (5). Aspiration of gastric contents is commonly accompanied by pulmonary exposure to oropharyngeal flora. Moreover, after acid aspiration, some critically ill patients need oxygen therapy with intratracheal intubation, which increases the incidence of nosocomial pneumonia (6). Once a patient suffers acid aspiration, the lungs are thus at increased risk of nosocomial infection (7).
We hypothesized that acid aspiration primes the host to enhance the inflammatory response to bacterial challenge after acid aspiration, which makes the patients susceptible to ARDS. The "two-hit phenomenon" suggests that an initial insult primes inflammatory cells such that a subsequent (otherwise innocuous) inflammatory insult causes an exaggerated response. There is some evidence in support of such a scen-ario: priming with endotoxin (8) or bacillus Calmette-Guérin (BCG) (9) increases the lung injury by a subsequent endotoxin challenge. These experimental models well explain the pathogenesis of ARDS induced by a pulmonary infection superimposed on systemic inflammatory response syndrome in which endotoxin translocation plays a significant role. Whether acid aspiration acts as an initial insult to prime the host still needs to be investigated.
Accumulating findings support the idea that protein tyrosine kinase (PTK)-dependent pathways play pivotal roles in the inflammatory cascade (10, 11). Tyrosyl phosphorylation is also reported to be required in the process of priming in neutrophils (12). We therefore hypothesized that acid instillation enhanced the inflammatory response to endotoxin through a PTK-dependent mechanism.
The purpose of the present study was to investigate the effect of acid instillation on the inflammatory response to the subsequent lipopolysaccharide (LPS) challenge in a rat model. We also studied the role of PTK in the inflammatory response to LPS after acid instillation using PTK inhibitors genistein and tyrphostin AG556 as a pharmacologic tool.
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METHODS |
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INTRODUCTION
METHODS
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Animals
Pathogen-free Wister rats weighing 300 ± 10 g were studied at 9 to 10 wk of age. All animals were housed in air-filtered, temperature-controlled units with access to food and water ad libitum. All experimental protocols were approved by the animal care committee and all experiments were done in conformity with the "Guiding Principles for Research Involving Animals" of Yokohama City University School of Medicine.
Reagents
Evan's blue was added to the hydrochloric acid (0.1 N; Wako Co., Osaka, Japan) to improve visualization of the site of instillation. LPS (055:B5 Escherichia coli; Sigma Chemical Co., St. Louis, MO) was dissolved in phosphate-buffered saline (PBS) and used at a concentration of 7.5 mg/ml. Genistein (4,5,7-trihydroxyisoflavone) was obtained from Sigma Chemical Co. and tyrphostin AG556 (N-[4-phenylbutyl]-3,4- dihydroxybenzylidene cyanoacetamide) was purchased from Calbiochem-Novabiochem Co. (La Jolla, CA). Genistein and tyrphostin AG556 dissolved in dimethyl sulfoxide (DMSO) were diluted to 1.5 mg/ml and 3.0 mg/ml respectively in PBS for injection. As a control the same final concentration of DMSO in PBS was used.
General Experimental Protocol
Rats were anesthetized with ether and placed in a 60° inclined position. A volume of 0.1 ml of hydrochloric acid (pH 1.0) or PBS was instilled to the lower segment of the right lung using a stainless steel animal feeding needle (18-gauge; Popper & Sons, Inc., New York, NY).
Twenty-two hours later, rats were reanesthetized with intraperitoneal sodium barbiturate (50 mg/kg). The trachea was exposed through an anterior neck incision and catheterized with 16-gauge tube. Through this tube, the rats were ventilated with a constant volume pump (model SN-480-7; Shinano Co., Tokyo, Japan) with an inspired oxygen fraction of 1.0, a tidal volume of 10 ml/kg at a rate of 60 cycles/min. The respiratory rate was adjusted to maintain arterial PCO2 between 35 and 45 mm Hg. Catheters of 24-gauge were inserted into the left jugular vein for drug infusion and the right carotid artery for blood sampling. Intravenous saline of 3 ml/h was administered to replace insensible water loss and volume lost by blood sampling. Rats were kept warm with homeothermic pad during the experiment.
Specific Experimental Protocol
The experimental protocol is depicted schematically in Figure 1. Fourteen animals were included in each experimental group. At the end of the observation period, thoracotomy was performed to confirm that acid had been instilled into the right lower lung segment. Wet to dry weight ratios were measured in seven rats in each group. Bronchoalveolar lavage (BAL) was performed in the other seven rats in each group.
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Group 1. A volume of 0.1 ml of PBS was instilled into the right lung of the control group. A volume of 1 ml of PBS containing DMSO was administered intraperitoneally at 23 h, and 0.1 ml of PBS was instilled into the lower segment of the left lung at 24 h.
Group 2. A volume of 0.1 ml of HCl was instilled into the right lung. PBS was administered at 23 and 24 h, as previously described.
Group 3. Same as Group 1, except 0.1 ml of LPS (2.5 mg/kg) rather than PBS was instilled into the left lung at 24 h.
Group 4. Same as Group 2, except 0.1 ml of LPS rather than PBS was instilled into the left lung at 24 h.
Group 5. HCl was instilled into the right lung. Twenty-three hours later, genistein (5.0 mg/kg) dissolved in 1.0 ml of PBS containing DMSO was injected intraperitoneally. At 24 h, LPS was instilled into the left lung.
Group 6. Same as Group 5, except tyrphostin AG556 (10 mg/kg) rather than genistein was administered intraperitoneally at 23 h.
Measurements
Arterial blood gas analysis. Arterial pH, PO2, and PCO2 were analyzed before the instillation of LPS or PBS, and then hourly thereafter. We used a 280 Blood Gas analyzer (Ciba-Corning Co., MA) with a sample volume of 0.3 ml.
Wet to dry weight ratio. The organ edema was assessed by wet to dry weight ratio. The right lung, left lung, heart, liver, kidneys, and the small intestine were harvested, weighted, dried in an oven at 60° C for 72 h and reweighed.
BAL and cell count. BAL of the whole left and whole right lungs
were performed in sequence while the other bronchus was clamped. For each lung, 3 ml of saline was lavaged 3 times through a tracheostomy tube. Fluid recovery was always above 85% and there was no
significant difference among the groups. BAL fluid (BALF) was centrifuged at 1,500 g for 10 min at 4° C and the supernatant was frozen at 
70° C until assay. The pellets were resuspended in 0.5 ml of PBS. After staining with gentian violet, the number of cells was counted using
a hemocytometer.
Cytokine assay. Four hours after the administration of LPS into
the left lung, rats were exsanguinated. Plasma was prepared in endo-toxin-free tubes containing ethylenediaminetetraacetic acid (EDTA)
and frozen at 70° C until assay. The concentration of tumor necrosis
factor-alpha (TNF-) in the plasma and in BALF was determined by
ELISA kit (TNF-, rat, ELISA system; Amersham International plc,
Amersham, UK). According to the manufacturer, there is no cross reactivity observed with rat interleukin-1 (IL-1) and IL-1
. The intra-assay and interassay coefficients of variance are less than 10%, and the
assay is sensitive to 10 pg/ml.
To quantitate cytokine-induced neutrophil chemoattractant (CINC)
in the BALF, we used an ELISA kit (IL-8 [rat GRO/CINC-1], rat,
ELISA system (Amersham International plc). The minimum detection level for rat GRO/CINC-1 was 4.7 pg/ml with the kit. There is no
cross reactivity with rat GRO/CINC-2, rat GRO/CINC-2, and rat
GRO/CINC-3 according to the manufacturer. The intra-assay and interassay coefficients of variance are 3.0 to 3.7% and 2.9 to 4.5%, respectively. All samples were analyzed in duplicate.
Myeloperoxidase (MPO) assay. One gram of blotted dry tissue was homogenized in 10 ml of 0.01 M potassium phosphate buffer (PPB, pH 7.4) containing 1.0 mM EDTA. Two milliliters of homogenate and 5.0 ml of 0.01 M PPB containing 1.0 mM EDTA were mixed gently then centrifuged at 10,000 g for 20 min at 4° C. The pellet was rehomogenized in 5.0 ml of 0.05 M PPB (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide. This suspension was freeze-thawed and sonicated with a Branson cell disrupter at 65 watt for 1 min. Aliquots of 0.1 ml were mixed with 0.79 ml of 0.08 M PPB (pH 5.4) and 0.1 ml of 16 mM tetramethylbenzidine dissolved in N,N-dimethylformamide at 37° C. After 2 min, 0.01 ml of 30 mM H2O2 was added. This solution was incubated for 3 min at 37° C, then 0.05 ml of 300 µg/ml catalase solution was added. This mixture was then diluted with 4.0 ml of 0.2 M sodium acetate (pH 3.0) and then centrifuged at 12,000 g for 10 min at 4° C. The supernatants were read in a spectrophotometer (UV-2000; Shimazu, Kyoto, Japan). One unit of MPO activity was defined arbitrarily as the amount of enzyme necessary to catalyze an increase in absorbance of 1.0 at 655 nm per minute at 37° C.
Nitric oxide (NO). NO was measured with an automated NO detector-high-performance liquid chromatography (HPLC) system
(ENO-20; Eicom, Kyoto, Japan). NO2
and NO3, the stable end
products of nitric oxide, were used as an indirect measure of NO.
Briefly, NO2 and NO3 in the plasma and in the BALF were separated by a reverse-phase separation column packed with polystyrene
polymer (NO-PACK; Eicom), and NO3 was reduced to NO2 in a
reduction column packed with copper-plated cadmium fillings (NO-RED; Eicom). NO2 was mixed with Griess reagent to form a purple azo
dye in a reaction coil. The separation and reduction column and the
reaction coil were placed in a column oven that was set at 35° C. The
absorbance of the color of the product dye at 540 nm was measured
by a flow-through spectrophotometer (NOD-10; Eicom). The mobile
phase, which was delivered by a pump at a rate of 0.33 ml/min, was
10% methanol containing 0.15 M NaCl, 0.15 M NH4Cl, and 0.5 g/L
4Na-EDTA. The Griess reagent, which was 1.25% HCl containing 5 g/L
sulfanilamide with 0.25 g/L N-naphthylethylenediamine, was delivered at
a rate of 0.1 ml/min. The contamination of NO2 and NO3 in PBS
used as BAL and the reliability of the reduction column were examined
in each sample.
Protein assay. The protein concentration of BALF was determined using the method of Bradford (13) as recommended by the protein kit manufacturer (Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA). Bovine albumin was used as a standard.
Endotoxin assay. The plasma endotoxin concentration was measured by the Limulus test using a commercially available kit (Endospec ES test TE; Seikagaku, Tokyo, Japan). Detection limit of this kit was 3.9 pg/ml (0.009 endotoxin unit [EU]/ml).
Statistical Analysis
All data are presented as mean ± SEM. One-way analysis of variance (ANOVA) with repeated measurements analysis was used to compare samples obtained at several time points from the same animals. One-way ANOVA (factorial) was used when comparing single groups. Fisher's post hoc test was used to determine which groups were significantly different. A p value less than 0.05 was considered statistically significant.
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RESULTS |
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To evaluate the contribution of acid exposure to subsequent susceptibility to LPS challenge, we instilled 0.1 ml of acid into the right lower segment of the lungs of experimental rats. LPS was then delivered to the left lung of some animals. A description of the treated and control animals is provided in Figure 1.
Changes in the Arterial Blood Gas
We initially evaluated whether the combination of acid plus LPS impedes oxygenation, a critical measure of lung function. Arterial PO2 values before the instillation of LPS or PBS into the left lung at 24 h did not differ significantly between groups, despite the prior administration of acid into the right lung of animals in Groups 2, 4, 5, and 6. After the introduction of either LPS or PBS into the left lung, there was a rapid decrease in arterial PO2 (Figure 2). In Groups 1-3, (PBS control, HCl-alone, and LPS-alone), the decrease in arterial PO2 was transient and arterial PO2 returned to baseline values within 2 h. In the HCl-LPS group, arterial PO2 continued to decrease throughout the observation period, reaching levels significantly below those of the other groups.
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p < 0.05 compared with
the HCl-LPS group.
The effect of HCl followed by LPS was significantly attenuated by intraperitoneal administration of genistein or tyrphostin AG556.
Analysis of the Pulmonary Edema
The fluid content of the lungs was monitored by comparing the wet-to-dry-weight ratio of lungs from rats in each group. Neither acid instillation to the right lung nor LPS instillation to the left lung alone increased wet-to-dry weight ratios when compared with those treated with PBS (Figure 3). In contrast, the wet-to-dry weight ratio of the right lung in the HCl-LPS group was significantly increased as compared with the control or LPS-alone group. This ratio was also elevated in the left lung, although this difference did not reach statistical significance. The PTK inhibitors genistein and tyrphostin AG556 attenuated the increase in wet-to-dry ratio in both lungs.
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We also analyzed the wet-to-dry-weight ratios of the other organs (including the heart, liver, kidneys, and small intestine), but found no significant difference between groups (data not shown).
Analysis of TNF- Production
TNF- is an important mediator of acid-induced and endotoxin-induced acute lung injury (14, 15). The concentration of immunoreactive TNF- in the BALF from the right lung of
the HCl-LPS group was significantly higher than that of any
other group (Figure 4). Indeed, TNF- was barely detectable
in the BALF of the control groups. Consistent with earlier results, the effect of HCl-LPS treatment was attenuated by genistein and tyrphostin AG556.
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In contrast, TNF- concentrations were dramatically increased in the left lung of both LPS-alone and HCl-LPS
treated groups. This effect was not attenuated by genistein or
tyrphostin AG556 treatment.
No significant differences in plasma TNF- were observed
among groups.
Analysis of Neutrophil Accumulation to the Lungs
To assess the degree of inflammation induced by the combination of acid plus LPS, we monitored the production of CINC, which is chemotactic for rat neutrophils (16), and the leukosequestration in the tissues. MPO activity was used as a tracer to quantitate polymorphonuclear sequestration in the tissues. This assay is more sensitive for detecting sequestered neutrophils than histology (17, 18).
A similar pattern as TNF- production was observed when
CINC and MPO concentrations were measured (Figures 5 and
6). In the right lung, the concentration of both of these agents
was selectively elevated in the HCl-LPS group; an effect that
was inhibited by genistein and tyrphostin AG556. In the left
lung, the concentration of CINC and MPO was significantly
increased in the LPS-alone and HCl-LPS groups, and this effect was not attenuated by genistein or tyrphostin AG556. Of
note, a selective increase in MPO concentration was also present in the small intestine of the HCl-LPS group.
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BAL Cellularity
The influx of inflammatory cells into the alveolar spaces was monitored by enumerating cells in BALF. The neutrophil count in the right lung of HCl-alone and LPS-alone groups was not significantly increased compared with controls (Figure 7). In the HCl-LPS group, the neutrophil count was significantly increased. The effect was attenuated by genistein and tyrphostin AG556.
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In the left lung, the acid instillation and subsequent intrabronchial LPS administration enhanced neutrophil mobilization. The effect was significantly suppressed but not totally eliminated by the PTK inhibitors.
The acid instillation followed by LPS challenge significantly increased macrophage mobilization to the alveolar spaces in the lungs, yet this effect was not eliminated by treatment with protein kinase inhibitors (data not shown).
NO
NO is a gaseous mediator of systemic inflammation and a potential mediator of acute lung injury (19). Concentration of plasma NO increases in pulmonary inflammation. In the LPS-alone or the HCl-alone group, the plasma concentration of NO was not increased compared with the control group (Figure 8). LPS instillation after the acid instillation was associated with significant increase in plasma NO, which was attenuated by genistein or tyrphostin AG556. The production of NO in the alveolar space of the lungs was not elevated as compared with the control in the LPS-alone group but significantly increased in the HCl-LPS group. Tyrphostin AG556 but not genistein significantly inhibited the increased response.
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Protein Concentration
Increased protein permeability across the endothelial and epithelial barriers of the lungs is the most fundamental physiologic characteristic of lung injury. We measured protein concentration in the BALF (Figure 9).
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Protein concentration of the right BALF in the LPS-alone group was not increased as compared with the control group. In the HCl-alone and HCl-LPS groups, the protein concentration increased, which was not attenuated by genistein or tyrphostin AG556.
In the left lung, the protein concentration of the BALF of the HCl-alone or the LPS-alone group was not different from the control. In the HCl-LPS group the protein concentration was significantly increased. Genistein and tyrphostin AG556 significantly inhibited the increase of protein permeability.
Endotoxin Concentration in the Plasma
To evaluate whether LPS instilled into the left lung reached the right lung through the systemic circulation, we measured plasma endotoxin concentration. In the control and HCl-alone groups, plasma endotoxin was undetectable. In LPS-instilled animals, trace plasma endotoxin was detected (less than 20 pg/ml: 0.05 EU/ml). There was no difference in plasma endotoxin concentrations between these groups (data not shown). Thus leakage of endotoxin could not account for the inflammatory response of rats of the HCl-LPS group.
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DISCUSSION |
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Results of this study indicated that localized exposures of the right lung to acid significantly increase the inflammatory response of the lungs induced by subsequent intrabronchial LPS challenge. Of particular importance, acid pretreatment significantly magnified and prolonged the decrease in arterial PO2 induced by intrabronchial LPS. The data also suggest that a PTK-dependent pathway is involved in this inflammatory response induced by acid instillation.
Acid aspiration is associated with biphasic lung injury (20). The direct injury caused by acid is a chemical burn and postulated to be short-lived, because the acid is rapidly neutralized. Subsequent recruitment and activation of neutrophils after acid aspiration is responsible for the indirect but greater pathology in the lungs and systemic organs (4).
With this experimental paradigm, we demonstrated the direct and indirect lung injury caused by acid instillation in the right-instilled lung as well as an indirect injury in the left contralateral lung. We then instilled LPS into the left lung to prove that the prior acid instillation had increased the left lung's susceptibility to this challenge.
It is reported that pulmonary infection is often diagnosed a few days after acid aspiration (5, 7). We instilled LPS 24 h after acid instillation to simulate this clinical scenario. It is also reported in animal experimental models that acid instillation increases permeability of the lungs as an indicator of pulmonary damage, which returns to control level some 15 h after acid instillation (20). In this study, we waited for 24 h after acid instillation until this biologic marker returned to control level so that we could evaluate the LPS-induced response.
We evaluated the effect of LPS 4 h after the instillation. Ulich and coworkers previously demonstrated that TNF messenger RNA (mRNA) peaks 2 to 4 h (15) and CINC peaks 4 h after the intratracheal instillation of LPS (21). Similarly, BAL
TNF- peaks at 3 h and BALF protein is increased for 5 h after endotoxin challenge (22). Thus we selected 4 h after LPS
instillation to evaluate the inflammatory response in the lungs.
In the right lung, the acid instillation significantly increased
TNF- and CINC, accumulation of neutrophils, and their emigration to the alveolar spaces in response to LPS challenge.
Prior acid instillation significantly increased the influx of neutrophils to the alveolar spaces and the permeability of protein
in the left lung. Wet to dry ratio, indicating pulmonary edema,
and NO production were increased. Oxygenation was impaired. These results indicate that the acid instillation increased the inflammatory response to LPS.
The findings of this study suggest that pathogenesis of ARDS after acid aspiration-induced lung injury can be explained through a two-hit model. In this model, an initial insult primes the host to generate an amplified response after a second insult. The initial hit (acid instillation in this study) predisposed the rats to mount an augmented inflammatory response to the second hit (LPS challenge). The same dose of LPS or HCl alone caused only a localized inflammatory response. The combination, however, generated a generalized inflammatory response detected in both lungs and in the small intestine.
We also examined whether PTK, which phosphorylates tyrosine residues, was involved in the enhancement of the inflammatory response to the intrabronchial LPS after acid instillation using PTK inhibitors as pharmacologic probes.
PTK inhibitors prevented the prolonged decrease in arterial PO2 induced by the intrabronchial LPS after acid instillation. In the right acid-instilled lung, the PTK inhibitors genistein or tyrphostin AG556 attenuated the increase of TNF-, CINC,
and neutrophil accumulation and mobilization to the airspaces.
In the left LPS-instilled lung, genistein or tyrphostin AG556
suppressed the increase of neutrophil emigration to the airspaces,
which was accompanied by the suppression of protein leakage.
PTK inhibitors did not attenuate the impairment of the alveolar blood barrier of the acid-instilled lung because acid instillation caused direct lung injury, increasing protein leakage in the instilled but not contralateral lung (23, 24).
These observations indicate that acid aspiration enhances the inflammatory response against LPS through the activation of tyrosine kinase cascades. Recent reports, including our observation, support the idea that phosphorylation of tyrosine residue plays a crucial role in the priming. It is reported in human neutrophils that a PTK-dependent pathway is already activated, i.e., tyrosine residue has been phosphorylated in the primed state (25, 26).
PTK is reported to be also involved in signal processing in
the cells, one of which is known as extracellular signal regulated kinase (ERK). Signals from LPS and most of the cytokines
including TNF- are in part transducted by PTK-dependent
pathways (27, 28). In this experiment, however, it is less possible
that genistein and tyrphostin AG556 directly inhibited LPS-induced or TNF--induced signal transduction and inflammatory
response because these PTK inhibitors did not suppress the localized inflammatory response such as production of TNF- and
CINC, and accumulation of neutrophils by LPS in the left lung.
The inflammatory response to endotoxin is compartmentalized when the alveolar capillary barrier of the lung is preserved (29). Yet, once the alveolar blood barrier is disturbed, cytokines are released into the circulation and promote inflammation at remote sites (30). It is conceivable that instilled endotoxin reached the contralateral lung through the systemic circulation and elicited the enhanced inflammatory response observed in our experiments. To evaluate this possibility, we measured the plasma concentration of endotoxin; we found that it was only slightly increased and that there was no significant difference among the LPS-instilled groups. This suggests that leaked LPS is not responsible for the augmented response observed in this study.
We demonstrated in this study that acid instillation primes the rat to augment the inflammatory response to endotoxin and that a two-hit model explains this phenomenon. In this study we examined the response to LPS. The response to live bacteria is different (31). We also demonstrated that a PTK-dependent mechanism is involved in the augmented response to LPS challenge after acid instillation.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Hiroshi Yamada, M.D., Department of Anesthesiology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-004, Japan.
(Received in original form July 13, 1999 and in revised form April 28, 2000).
Acknowledgments: The authors wish to thank Dr. Dennis M. Klinman for reviewing this manuscript. They also thank Dr. Kiyoyasu Kurahashi for his technical assistance.
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References |
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ABSTRACT
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METHODS
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REFERENCES
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