Differential NF-kappa B activation after intratracheal endotoxin

Timothy S. Blackwell, Lisa H. Lancaster, Thomas R. Blackwell, Annapurna Venkatakrishnan, and John W. Christman

Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville 37232-2650; and Department of Medicine, Department of Veterans Affairs, Nashville, Tennessee 37212


    ABSTRACT
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ABSTRACT
INTRODUCTION
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We examined the relationship between nuclear factor (NF)-kappa B DNA binding activity, cytokine gene expression, and neutrophilic alveolitis in rats after intratracheal (IT) instillation of endotoxin [lipopolysaccharide (LPS)]. NF-kappa B activation in lung tissue mirrored neutrophilic alveolitis after IT LPS instillation, with NF-kappa B activation and neutrophilic influx beginning 2 h after IT LPS doses of 0.01 mg/kg or greater. In lung lavage fluid cells, however, transient NF-kappa B activation was present in alveolar macrophages by 15 min after IT LPS instillation, followed by a second peak of NF-kappa B activation corresponding to the onset on neutrophilic alveolitis. For cytokines thought to be NF-kappa B dependent, two different patterns of mRNA expression were found. Interleukin (IL)-1alpha , IL-1beta , and tumor necrosis factor-alpha showed increased mRNA by 30 min after IT LPS instillation, but IL-6- and cytokine-induced neutrophil chemoattractant mRNAs were not substantially increased until 2 h after IT LPS instillation. Therefore, IT LPS causes differential NF-kappa B activation in air space cells and lung tissue, which likely determines production of key cytokines and directs the evolution of neutrophilic alveolitis.

nuclear factor-kappa B; lung; neutrophil; inflammation; chemokine; macrophage


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PRODUCTION OF ACUTE inflammatory cytokines and influx of activated neutrophils into the lungs are thought to be important in the pathogenesis of lung injury in acute respiratory distress syndrome, which can occur as the result of direct pulmonary insults including bacterial pneumonia, gastric acid aspiration, and inhalation of toxic gases. In animal models, Ulich et al. (20) and other investigators (8, 11, 27) have shown that intratracheal (IT) administration of bacterial endotoxin [lipopolysaccharide (LPS)] causes both lung cytokine production and neutrophilic influx. In this study, we examined the relationship between cytokine gene expression, neutrophilic alveolitis, and activation of the transcription factor complex nuclear factor (NF)-kappa B in rats treated with IT LPS.

NF-kappa B is a ubiquitous transcription factor that exists as a dimer of two Rel proteins. In unstimulated cells, NF-kappa B is bound to an inhibitor (Ikappa B) that anchors the protein complex in the cell cytoplasm. Inflammatory stimuli, including LPS, tumor necrosis factor (TNF)-alpha , interleukin (IL)-1, viral proteins, and mitogens, cause phosphorylation, ubiquitinization, and degradation of Ikappa B. Liberated NF-kappa B then translocates to the nucleus, binds to specific sites on DNA, and enhances the expression of a variety of genes that are important for promoting acute inflammation (4, 13). Mediators of inflammation that are transcriptionally regulated by NF-kappa B in vitro include cytokines (TNF-alpha , IL-1beta , and IL-6), C-X-C chemokines [IL-8; GROalpha , -beta , and -gamma ; macrophage inflammatory protein-2; and cytokine-induced neutrophil chemoattractant (CINC)], and enzymes (cyclooxygenase-2 and inducible nitric oxide synthase) (4, 13).

Our hypothesis in these studies was that IT LPS would induce specific patterns of NF-kappa B activation in lung lavage fluid cells and lung tissue and that NF-kappa B activation would correlate with NF-kappa B-dependent cytokine gene expression and neutrophilic alveolitis. We anticipated that the earliest event in the development of neutrophilic lung inflammation would be increased nuclear binding activity of NF-kappa B proteins in alveolar macrophages because these cells are thought to play a role in the initiation of the inflammatory cascade. We evaluated the dose requirements of LPS and the timing of NF-kappa B activation in whole lung tissue and lung lavage fluid cells and found a differential pattern of NF-kappa B activation. We compared the pattern of NF-kappa B binding activity to neutrophilic alveolitis, gene expression of a panel of acute inflammatory cytokines, and the rat neutrophil chemoattractant chemokine CINC. Our findings show that NF-kappa B activation in the lung after IT LPS instillation correlates with cytokine mRNA expression and neutrophilic alveolitis, supporting the idea that NF-kappa B activation is a pivotal event in the generation of neutrophilic lung inflammation. Interestingly, a burst of NF-kappa B binding activity in alveolar macrophages precedes neutrophilic inflammation and correlates with whole lung gene expression of TNF-alpha and IL-1, consistent with the hypothesis that alveolar macrophage activation triggers the inflammatory process.


    METHODS
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INTRODUCTION
METHODS
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Animal model. Male Sprague-Dawley rats weighing between 200 and 300 g were used in all experiments. Escherichia coli LPS (serotype 055:B5; Sigma, St. Louis, MO) was given by a single IT injection in doses of 0-6 mg/kg. For IT injections, the rats were anesthetized with ketamine and xylazine, and LPS (diluted in sterile physiological saline) was instilled directly into the trachea in a total volume of 300-400 µl. The rats were asphyxiated with carbon dioxide, the tracheae were cannulated after death, and the lungs were lavaged in situ with sterile pyrogen-free physiological saline. Saline was instilled in two 5-ml aliquots and gently withdrawn with a 5-ml syringe. For other studies, the lungs were removed, quickly frozen in liquid nitrogen, and stored at -70°C.

Cell counts and differentials. Lung lavage fluid was centrifuged at 500 g for 10 min to separate cells from supernatant. Pelleted cells were resuspended in a small amount of physiological saline with 1% BSA. Total cell counts were determined on a grid hemacytometer. Differential cell counts were enumerated on cytocentrifuge slides that were stained with a modified Wright stain (Diff-Quik) by counting 400-600 cells in cross section.

Nuclear protein extractions for lavage fluid cells and lung tissue. Nuclear proteins from lavage fluid cells and from tissue were prepared as previously described (1, 2). For lavage fluid cell nuclear extracts, lavage fluid cells from two different rats at each time point were combined to ensure adequate nuclear protein yields.

Electrophoretic mobility shift assays. An oligonucleotide probe containing a consensus NF-kappa B motif, 5'-GATCGAGGGGACTTTCCCTAGC-3' (Stratagene, La Jolla, CA), was used in these studies. End labeling was accomplished by treatment with T4 kinase in the presence of [32P]ATP. Labeled oligonucleotide was column purified on a Sephadex G-25M column (Pharmacia Biotech, Piscataway, NJ). Labeled double-stranded probe (~100,000 counts/min) was added to 5 µg of nuclear protein in the presence of binding buffer (Stratagene). This mixture was incubated at 25°C for 20 min and separated by electrophoresis on a 6% polyacrylamide gel in 1× Tris-borate-EDTA buffer. The gels were vacuum-dried and subjected to autoradiography. Cold competition was done by adding 50 ng of specific unlabeled double-stranded probe to the reaction mixture. The specificity of the DNA binding was confirmed by adding an excess of an unrelated nonspecific DNA sequence, 5'-GATCGAATGCAAATCACTAGCT-3', that contains an Oct-1 binding motif. On some gels, supershift assays were done with polyclonal antibodies to Rel A p65 obtained from Santa Cruz Biotechnology. Two micrograms of the antibody were added to the reaction mixture, and the samples were incubated at 25°C for 1 h before gel loading.

Tissue RNA extractions. Total RNA was purified with a modification of the method of Chirgwin et al. (6). Frozen lung tissue was mixed with 1 ml of TRI REAGENT (Molecular Research Center, Cincinnati, OH) and ground in a tissue homogenizer. The samples were transferred to 1.5-ml Eppendorf tubes, and RNA was extracted with phenol-chloroform and precipitated with isopropanol. The RNA pellet was then washed with 75% ethanol, air-dried, and dissolved in 50-100 µl of 30% formamide-10% formaldehyde. Total RNA was quantitated by determining the light absorbance at 260 nm.

Northern blots. Total RNA was separated by electrophoresis on a 1% agarose-formaldehyde gel, transblotted to nitrocellulose, and fixed with ultraviolet light (Stratagene 1800). The filters were prehybridized, then hybridized with a specific 32P end-labeled 30-base oligonucleotide probe for CINC with the sequence 5'-GCGGCATCACCTTCAAACTCTGGATGTTCT-3', which is complementary to nucleotides 134-164 of CINC cDNA. Blots were high-stringency washed, and CINC mRNA was detected by autoradiography in the presence of intensifying screens.

RNase protection assay. RNase protection assays (RPAs) were done with the RiboQuant multiprobe RPA system (PharMingen, San Diego, CA). A rat cytokine template set (rCK-1) was used. The probes were made and radiolabeled by adding [alpha -32P]UTP to a reaction mixture including template, transcription buffer, and T7 polymerase. After incubation, DNase was added, and the RNA probes were purified by phenol-chloroform extraction followed by ethanol precipitation. The probes were resuspended in hybridization buffer and diluted to 4 × 105 counts · min-1 · µl-1. Twenty micrograms of total RNA from the lung were dried in a vacuum evaporator centrifuge and resuspended in 8 µl of hybridization buffer. Two micrograms of labeled probe were added to each sample, which was then heated to 90°C and incubated overnight at 56°C. RNase was then added, followed by proteinase K. Resultant RNase digests were then phenol-chloroform extracted and precipitated with ethanol. Loading dye was added to the dried pellets, and the samples were heated briefly to 90°C. A 5% polyacrylamide gel was then run, dried, and subjected to autoradiography.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Initially, we determined the time course and dose response for IT LPS induction of neutrophilic alveolitis and NF-kappa B binding activity in nuclear extracts of lung tissue (Figs. 1 and 2). Figure 1A shows the percentage of alveolar macrophages and neutrophils in lung lavage fluid 0, 0.5, 1, 2, 4, and 6 h after treatment with IT LPS at 6.0 mg/kg, a dose that causes neutrophilic lung inflammation after intraperitoneal injection of LPS (5). In control lungs and up to 1 h after IT LPS instillation, >96% of lavage fluid cells were alveolar macrophages. By 2 h, neutrophils accounted for 65% of lavage fluid cells, and this percentage increased to 82% by 4 h and to 94.5% by 6 h after IT LPS instillation. The total number of lavage fluid neutrophils and macrophages is reported in Fig. 1B. As shown, there was a fivefold rise in the total number of lavage fluid cells by 6 h, which was entirely due to a massive increase in the number of neutrophils.




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Fig. 1.   Time course for bronchoalveolar lavage (BAL) fluid cell counts after treatment with intratracheal (IT) lipopolysaccharide (LPS). Rats were killed 0, 0.5, 1, 2, 4, and 6 h after IT treatment with LPS at 6 mg/kg. Percent (A) and total (B) alveolar macrophages and neutrophils in BAL are shown as means from 2 rats at each time point (total 12 rats). C: nuclear factor (NF)-kappa B activation in lung tissue is shown by electrophoretic mobility shift assay (EMSA). Each lane represents a lung nuclear protein extract from a different rat at indicated times (nos. on top in h). Band B represents p50 homodimers because this band shifts with antibodies to p50 but not to Rel A (data not shown). Band C represents Rel A/p50 heterodimers that migrate below p50 homodimer band because of ex vivo COOH-terminal truncation of Rel A (see text).

Using lung tissue from these rats, we next determined the effect of IT LPS on NF-kappa B binding activity. Figure 1C shows the time course for the appearance of NF-kappa B binding activity in nuclear protein extracts from lung tissue. With electrophoretic mobility shift assays from rat lung tissue, we can detect up to three bands, which we labeled bands A, B, and C. These bands have been identified by supershifted electrophoretic mobility shift assays with antibodies to the NH2 terminus and COOH terminus of Rel A p65 and to p50 (1, 2) (data not shown). Band A contains intact Rel A/p50 heterodimers (binds to all three antibodies), band B contains p50 homodimers (binds to only the p50 antibody), and band C contains Rel A/p50 heterodimers in which the Rel A is truncated at the COOH terminus by an ex vivo event during nuclear protein extraction [binds to p50 and the NH2-terminal Rel A antibody but not to the COOH-terminal Rel A antibody (1, 2); therefore, bands A and C contain "classic" NF-kappa B (Rel A/p50)]. Bands containing Rel A are of particular interest because Rel A, but not p50, contains a COOH-terminal transcription activation domain (12). In Figure 1C, only bands B and C were identified. Lung Rel A/p50 (band C) binding activity was minimal at 30 min and 1 h after IT LPS instillation but was markedly upregulated by 2 h after treatment with IT LPS and sustained to 6 h. The p50 homodimer (band B) was present in untreated rats and remained detectable throughout the time course. Therefore, in lung tissue, NF-kappa B (Rel A/p50) binding activity correlates directly with neutrophil influx into the alveolar space, which implies that the detected NF-kappa B binding activity could be due, at least in part, to NF-kappa B activation in the neutrophils themselves.

After completing time-course studies, we investigated the dose-response characteristics of neutrophilic alveolitis after IT LPS instillation. Figure 2A shows the percentage of alveolar macrophages and neutrophils in lung lavage fluid 4 h after treatment with IT LPS (0, 0.001, 0.01, 0.1, 1.0, and 6.0 mg/kg). The threshold LPS dose for significant induction of neutrophilic influx into the lung at 4 h was found to be 0.01 mg/kg. IT administration of physiological pyrogen-free saline, the vehicle, was not associated with the appearance of neutrophils in lung lavage fluid at the 4-h time point. A dose of 0.001 mg/kg of LPS produced 11 ± 8% neutrophils in lavage fluid, but a dose of 0.01 mg/kg of LPS produced 62 ± 4% neutrophils in lavage fluid at 4 h. IT LPS at 0.1, 1.0, and 6 mg/kg produced 61 ± 10, 71 ± 8, and 78 ± 3% neutrophils, respectively, at 4 h. Figure 2B shows the dose-response relationship between IT LPS dose (0.001 and 0.01 mg/kg) and NF-kappa B binding activity in lung tissue nuclear protein extracts. In Fig. 2B, both bands A and C were identified. Band A (intact Rel A/p50) was faintly detected in lung samples from rats treated with 0.001 mg/kg of IT LPS (Fig. 2B, lanes 1-3) but was not seen in samples from rats treated only with the normal saline vehicle (data not shown). In contrast, both bands A and C were substantially upregulated in rat lungs after treatment with 0.01 mg/kg of IT LPS (Fig. 2B, lanes 4-6). Similar NF-kappa B binding activity was seen with larger doses of IT LPS (data not shown). These data, together with our initial studies, show that activation of NF-kappa B in lung tissue mirrors the influx of neutrophils into lavagable air spaces after IT LPS instillation.



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Fig. 2.   A: effect of various IT LPS doses on BAL cell differential counts was determined 4 h after IT LPS instillation. Values are means ± SE; n = 3 rats at each LPS dose. B: EMSA with lung tissue nuclear protein extracts showing effect of IT LPS at indicated doses (nos. on top in mg/kg) on activation of NF-kappa B (Rel A/p50 heterodimers; bands A and C) at 4-h time point.

To better identify which cells were responsible for the measured NF-kappa B activation in the lungs after IT LPS instillation, we measured NF-kappa B binding activity in nuclear extracts of lung lavage fluid cells after IT LPS instillation. We performed lung lavages with a total of 35 ml of sterile saline, combined the lavage fluid cells from two different rats in each treatment group, and extracted nuclear proteins. Figure 3A shows the differential cell counts from an experiment in which 0.1 mg/kg of LPS was given intratracheally and the lungs were lavaged at various time points. In this experiment, there were few (<10%) neutrophils in the lavage fluid from 0 to 2 h after IT LPS instillation, and 77% neutrophils were present at 4 h. In the lavage fluid cells, NF-kappa B binding activity did not directly correlate with neutrophilic influx (Fig. 3B). In contrast to lung tissue NF-kappa B binding activity, lavage fluid cells exhibited two peaks of activity. There was an initial increase in NF-kappa B binding activity 15 min after IT LPS instillation (Fig. 3B, lane 2) that was diminished by 30 min and remained low until 4 h, when a second peak of NF-kappa B activity was found (Fig. 3B, lane 6). The initial peak of NF-kappa B binding activity appears to be in alveolar macrophages because >90% of lavage fluid cells were macrophages at that time point. No increase in NF-kappa B binding activity was found in whole lung nuclear protein extracts 15 min after IT LPS instillation (data not shown). At 4 h, it is likely that the detectable NF-kappa B binding activity was from neutrophils because these cells accounted for 77% of lavagable cells at this time point. Similar results were obtained when this experiment was repeated with 6 mg/kg of IT LPS except that the second peak of lavage fluid cell NF-kappa B binding activity and neutrophilic alveolitis were present 2 h after 6 mg/kg of IT LPS (data not shown). Figure 3B also shows cold and nonspecific competition (lanes 7 and 8, respectively), indicating specific nuclear protein binding to the NF-kappa B motif because most of the band is eliminated by adding excess unlabeled specific (cold) probe, but the band is not eliminated by adding the same amount of nonspecific probe. Although NF-kappa B bands A-C are not well separated in Fig. 3B, lane 9 shows that the band contains mostly Rel A p65 because antibodies to this NF-kappa B component result in diminution of this band. In summary, these data show that IT LPS results in early activation of NF-kappa B in alveolar macrophages that is short-lived, followed by a subsequent peak of NF-kappa B activity in lung tissue and in invading neutrophils that mirrors the alveolar neutrophilic influx. It seems unlikely that the NF-kappa B binding activity seen at later time points in entirely due to neutrophil NF-kappa B activation, but neutrophil NF-kappa B activation may lead to a positive feedback loop where NF-kappa B-dependent inflammatory mediator production may lead to further neutrophil influx and activation.



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Fig. 3.   A: time course for BAL cell counts after treatment with IT LPS at 0.1 mg/kg. Values are means from 2 rats at each time point. B: NF-kappa B activation in BAL cells [combined from 2 rats at each time point (nos. at top in h)]. Specificity of nuclear protein binding to NF-kappa B motif is shown by cold (C) and nonspecific (NS) competition (lanes 7 and 8, respectively). Addition of 50 ng of unlabeled NF-kappa B probe (C) to 4-h sample (lane 7) diminished band, but addition of same amount of unrelated, NS probe did not (lane 8). Band is shown to contain Rel A p65 because addition of NH2-terminal Rel A antibodies (labeled p65; lane 9) decreased intensity of this band.

We used a multiprobe RPA and Northern blots to correlate NF-kappa B binding activity with cytokine gene expression in the lung after IT LPS instillation. Figure 4 shows steady-state mRNA levels for a variety of cytokines after treatment with IT LPS (6 mg/kg) with RPA. After treatment with IT LPS, expression of IL-1alpha mRNA, IL-1beta mRNA, and TNF-alpha mRNA was apparent by 30 min and persisted to 6 h. Subtle expression of IL-6 mRNA was evident by 1 h, with more intense expression at 2, 4, and 6 h. TNF-beta , IL-2 to -5 and -10, and interferon-gamma mRNAs were not detectable in lung tissue after IT LPS treatment. L32 and glyceraldehyde-3-phosphate dehydrogenase are products of constitutive genes that are illustrated to show equal loading of RNA. In addition to this cytokine panel, we measured mRNA levels of the rat C-X-C chemokine CINC, which Blackwell and colleagues (1, 3, 5) and others (7, 9, 18) have shown to be an important neutrophil chemoattractant in the rat.


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Fig. 4.   Ribonuclease protection assay from rat lung tissue total RNA after treatment with IT LPS. Rats were treated with IT LPS (6.0 mg/kg), and lung tissue was harvested at indicated times (nos. at top in h) after treatment. Each lane represents results from a single animal. Position of detected cytokines [interleukin (IL)-1alpha , IL-1beta , IL-6, and tumor necrosis factor (TNF)-alpha ] and 2 constitutively expressed mRNAs, L32 and glyceraldehyde-3-dehydrogenase (GAPDH), is indicated. TNF-beta , IL-2 to -5 and -10, and interferon-gamma were not detectable in lung tissue after IT LPS treatment.

CINC mRNA expression as assessed by Northern blot is shown in Fig. 5. The kinetics of CINC gene expression in lung tissue (Fig. 5A) were similar to those of IL-6. CINC mRNA was upregulated 2 h after IT LPS instillation and persisted until 6 h after treatment with IT LPS, with minimal detectable CINC mRNA before 2 h. In dose-response studies (Fig. 5B), CINC mRNA was increased 4 h after doses of IT LPS ranging from 0.01 to 1 mg/kg but was only minimally detected in the lungs of one of two rats after treatment with 0.001 mg/kg of IT LPS.



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Fig. 5.   Northern blot of time course (A) and dose-response relationship (B) between IT LPS and expression of cytokine-induced neutrophil chemoattractant (CINC) mRNA. A: rats were treated with IT LPS (6.0 mg/kg), and total RNA was extracted from lung tissue at indicated times (nos. on top in h) after treatment. B: rats were treated with various IT LPS doses (nos. on top in mg/kg), and total RNA was extracted from lung tissue 4 h after treatment.

In these studies, expression of non-NF-kappa B-dependent cytokines (IL-3 to -5 and -10 and interferon-gamma ) was not induced in lung tissue by IT LPS. Two potentially NF-kappa B-dependent cytokines, TNF-beta and IL-2, were not detectable. Other cytokines thought to be NF-kappa B dependent showed two different patterns. First, CINC and IL-6 gene expression in lung tissue correlated well with both lung tissue NF-kappa B binding activity and neutrophilic alveolitis after IT injection of LPS. Second, IL-1alpha , IL-1beta , and TNF-alpha showed increased expression by 30 min after IT LPS instillation, before NF-kappa B binding activity in lung tissue but consistent with alveolar macrophage NF-kappa B activation, suggesting alveolar macrophages as a major source of these cytokines.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Our studies, like those of Ulich and colleagues (17-19, 21-24) and other investigators (16, 25, 26), show that IT administration of LPS is associated with lung cytokine production and neutrophilic influx. In addition, we have shown that IT LPS results in an early and intense neutrophilic influx that is closely associated with NF-kappa B binding activity in lung tissue. Both time-course and dose-response studies showed a close relationship between lung NF-kappa B binding activity, CINC gene expression, and neutrophilic alveolitis. We also discovered that the gene expression of several NF-kappa B-dependent cytokines in lung tissue was increased after IT LPS instillation and that the production of early proinflammatory cytokines, TNF-alpha , IL-1alpha , and IL-1beta , preceded peak NF-kappa B binding activity in lung tissue. Increased mRNA of these cytokines correlated much better with the early NF-kappa B binding activity that was identified in alveolar macrophages. Because alveolar macrophages are known to produce all three of these cytokines (15), it is likely that macrophages are a major source of these cytokines after IT instillation of LPS. This early production of cytokines (IL-1 and TNF-alpha ) may be an early response by alveolar macrophages that initiates the cytokine cascade and signals other cells in the lung to produce "distal" cytokines such as CINC and other chemokines that are important for signaling neutrophilic immigration. Lung epithelial cells, for example, are known to produce IL-8 in culture at much higher concentrations in response to TNF-alpha and IL-1 than in response to LPS (14).

Another interesting finding of the present study is the late appearance of NF-kappa B DNA binding activity in lavage fluid cells, corresponding to the time of neutrophil predominance in the lavage fluid cell population. Although neutrophils previously have been shown to activate NF-kappa B and produce inflammatory mediators (10), our results suggest that neutrophils in this model use NF-kappa B-dependent mechanisms to produce inflammatory mediators and amplify the inflammatory response. In combination, these data suggest that a complex pattern of cellular NF-kappa B activation and cytokine production coordinates neutrophilic alveolitis after IT LPS instillation.

Further insight into the systemic and local mechanisms of acute pulmonary inflammation via direct and indirect inflammatory stimuli may help to clarify the sequence of events leading to neutrophilic lung inflammation and lung injury. Key control points in the inflammatory cascade, such as NF-kappa B, may provide targets for intervention to modulate inflammation by local action in the lungs, targeting specific cell types or, systemically, targeting the balance between lung and systemic inflammatory mediator production. Our data suggest that agents that prevent activation of NF-kappa B in alveolar macrophages, if given early, could prevent propagation of the inflammatory cascade. In contrast, agents given later that have a broader cellular target in the lung could decrease the intensity of neutrophilic inflammation by preventing more distal elements of the inflammatory cascade.


    ACKNOWLEDGEMENTS

This work was supported by the Department of Veterans Affairs; a Parker B. Francis Foundation Fellowship in Pulmonary Research; The American Lung Association, and National Heart, Lung, and Blood Institute Grant HL-07123.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. S. Blackwell, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt Univ. School of Medicine, T-1217 MCN, Nashville, TN 27232-2650 (E-mail: timothy.blackwell{at}mcmail.vanderbilt.edu).

Received 20 January 1999; accepted in final form 26 May 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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19.   Ulich, T. R., S. C. Howard, D. G. Remick, E. S. Yi, T. Collins, K. Guo, S. Yin, J. L. Keene, J. J. Schmuke, and C. N. Steininger. Intratracheal administration of endotoxin and cytokines. VIII. LPS induces E-selectin and soluble E-selectin inhibit acute inflammation. Inflammation 18: 389-398, 1994[Medline].

20.   Ulich, T. R., L. R. Watson, S. M. Yin, K. Z. Guo, P. Wang, H. Thang, and J. del Castillo. The intratracheal administration of endotoxin and cytokines. I. Characterization of LPS-induced IL-1, TNF mRNA expression and the LPS-, IL-1- and TNF-induced inflammatory infiltrate. Am. J. Pathol. 138: 1485-1496, 1991[Abstract].

21.   Ulich, T. R., E. S. Yi, S. Yin, C. Smith, and D. Remick. Intratracheal administration of endotoxin and cytokines. VII. The soluble interleukin-1 receptor and the soluble tumor necrosis factor receptor II (p80) inhibit acute inflammation. Clin. Immunol. Immunopathol. 72: 137-140, 1994[Medline].

22.   Ulich, T. R., S. M. Yin, K. Z. Guo, J. del Castillo, S. P. Eisenberg, and R. C. Thompson. The intratracheal administration of endotoxin and cytokines. III. The interleukin-1 (IL-1) receptor antagonist inhibits endotoxin and IL-1 induced acute inflammation. Am. J. Pathol. 138: 521-524, 1991[Abstract].

23.   Ulich, T. R., S. Yin, K. Guo, E. S. Yi, D. Remick, and J. del Castillo. Intratracheal injection of endotoxin and cytokines. II. Interleukin-6 and transforming growth factor beta inhibit acute inflammation. Am. J. Pathol. 138: 1097-1101, 1991[Abstract].

24.   Ulich, T. R., S. Yin, D. G. Remick, D. Russell, S. P. Eisenberg, and T. Kohno. Intratracheal administration of endotoxin and cytokines. IV. The soluble tumor necrosis factor receptor type I inhibits acute inflammation. Am. J. Pathol. 142: 1335-1338, 1993[Abstract].

25.   Wesselius, L. J., I. M. Smirnov, A. R. O'Brien-Ladner, and M. E. Nelson. Synergism of intratracheally administered tumor necrosis factor with interleukin-1 in the induction of lung edema in rats. J. Lab. Clin. Med. 125: 618-625, 1995[Medline].

26.   Yi, E. S., D. G. Remick, Y. Lim, W. Tang, C. E. Nadzienko, A. Bedoya, S. Yin, and T. R. Ulich. The intratracheal administration of endotoxin. X. Dexamethasone down regulates neutrophil emigration and cytokine expression in vivo. Inflammation 20: 165-175, 1996[Medline].

27.   Yi, E. S., and T. R. Ulich. Endotoxin, interleukin-1, and tumor necrosis factor cause neutrophil-dependent microvascular leakage in postcapillary venules. Am. J. Pathol. 149: 659-663, 1992.


Am J Physiol Lung Cell Mol Physiol 277(4):L823-L830
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