Introduction

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Neutrophil recruitment and activation is a central component in the pathophysiology of acute lung injury (ALI) (1). In lung injury models, the mechanism by which neutrophils are recruited into the lung has been linked to requirements for proinflammatory cytokines (e.g., tumor necrosis factor [TNF]-) (2). TNF- increases expression of endothelial cell adhesion molecules that mediate anchoring of neutrophils to endothelial cells. Neutrophils then migrate into the lung parenchyma in response to chemotactic cytokines (chemokines). In humans, the principle chemoattractant and activator of neutrophils is interleukin (IL)-8 (3). IL-8 plays a central role in the pathophysiology of ALI. Bronchial alveolar lavage fluids of patients with ALI have consistently demonstrated increased levels of IL-8 and this is associated with increased mortality (4). Inhibiting IL-8 activity, or the activity of IL-8 homologues, confers protection in animal models of ALI (7). Collectively, these observations suggest that modifying IL-8 gene expression may have a beneficial effect in patients with ALI.

IL-8 gene expression is regulated in part by the transcription factor nuclear factor (NF)-B (11). NF-B normally resides in the cytoplasm bound to its inhibitory protein, IB (12, 13). In response to a variety of proinflammatory signals, IB is rapidly degraded by a phosphorylation- dependent and ubiquitination-dependent mechanism. Rapid degradation of IB allows for nuclear translocation of NF-B where it directs the expression of target genes such as IL-8.

Geldanamycin is a benzoquinone ansamycin that has been variably described as a tyrosine kinase inhibitor (14), an inhibitor of heat-shock protein 90 (15, 16), an antitumor agent (17), and most recently, an inducer of heat-shock proteins (18, 19). In vivo administration of geldanamycin attenuates lung inflammation and ALI in animal models (J. F. Pittet and W. J. Welch, unpublished observations), thereby suggesting that geldanamycin also has potent anti-inflammatory effects. Accordingly, in the current experiments we determined the effect of geldanamycin on in vitro IL-8 gene expression and on activation of the NF-B pathway in cultured human respiratory epithelial cells.

	Materials and Methods

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Cell Culture

All experiments involved A549 cells (American Type Culture Collection, Bethesda, MD), a human lung adenocarcinoma cell line representative of distal respiratory epithelium. These cells were previously demonstrated to be a useful model for studying in vitro IL-8 gene regulation in that they inducibly express the IL-8 gene in response to proinflammatory stimuli (20, 21). Cells were maintained in a room air/5% CO₂ incubator at 37°C using Dulbecco's modified Eagle's medium (GIBCO BRL, Grand Island, NY) containing 8% fetal bovine serum and penicillin/streptomycin (GIBCO BRL).

Experimental Conditions

IL-8 gene expression was induced by treating cells with human TNF- (Boehringer Mannheim, Indianapolis, IN) at a concentration of 2 ng/ml. One group of cells was treated with geldanamycin (1 µg/ml; working dose determined in preliminary experiments; J. F. Pittet, unpublished observations) for 1 h before incubation with TNF-. Geldanamycin stock solutions were prepared in dimethyl sulfoxide (DMSO) such that the final concentration in culture was 0.1% DMSO. Cells not treated with geldanamycin were preincubated in medium containing 0.1% DMSO.

IL-8 Enzyme-Linked Immunosorbent Assay

Immunoreactive IL-8 concentrations were measured in the medium of treated cells using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) (Biosource, Camarillo, CA). All procedures were performed as recommended by the manufacturer.

Northern Blot Analysis

Total cellular RNA was recovered using the Trizol reagent (GIBCO BRL). RNA was quantified by spectrophotometry (260 nM) and 15 µg of total RNA per condition underwent electrophoresis on 1% agarose gel containing 3% formaldehyde. Ethidium bromide staining and brief ultraviolet (UV) illumination confirmed integrity of the RNA after electrophoresis. RNAs were transferred to Nylon membranes (Micro Separations Inc., Westboro, MA) and UV auto-crosslinked (UV Stratalinker 1800; Stratagene, La Jolla, CA). After a 4-h prehybridization at 42°C, membranes were hybridized overnight with a radiolabeled human IL-8 complementary DNA (cDNA) probe (20). The cDNA was labeled with -[³²P]deoxycytidine triphosphate (specific activity, 3,000 Ci/mM; New England Nuclear Research Products, Boston, MA) by random priming (Pharmacia, Piscataway, NJ). The hybridized filters were serially washed at 53°C using 2× sodium chloride/ 0.1% sodium dodecyl sulfate (SDS) and 25 mM NaHPO₄/1 mM ethylenediaminetetraacetic acid (EDTA)/0.1% SDS solutions. After washing, exposure was carried out overnight and analyzed using a PhosphorImager screen and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Transient Transfections and Luciferase Assays

To measure IL-8 promoter activity, one group of cells was transiently transfected with a plasmid containing the 200 bp 5' flanking region of the IL-8 gene cloned into a luciferase reporter plasmid (pGL2; Promega, Madison, WI) such that the IL-8 promoter region drives inducible expression of luciferase (20). To measure NF-B activation, one group of cells was transiently transfected with a plasmid in which the luciferase gene was driven by three tandem NF-B binding motifs, followed by a minimal interferon- promoter (3× NF-B-Luc, a kind gift of Dr. Roland M. Schmid, University of Ulm, Ulm, Germany). This plasmid was previously demonstrated to be a sensitive tool to specifically evaluate NF-B activation (22).

Cells were transfected in duplicate, in six-well plates, at a density of 300,000 cells per well by incubation with cationic liposomes (Lipofectin; GIBCO BRL) for 5 h in OptiMEM (GIBCO BRL). After 5 h, OptiMEM medium was removed and regular medium was added to the transfected cells and incubated overnight. After exposing the cells to the experimental conditions, the cellular proteins were extracted and analyzed for luciferase activity according to the manufacturer's instructions (Promega) using a Berthhold AutoLumant LB953 luminometer. Luciferase activity was corrected for total cellular protein and reported as fold induction over the control cells (cells that were transfected and treated with medium alone).

Nuclear Protein Extraction

After exposing cells to the experimental conditions, nuclear protein extraction was performed on ice with ice-cold reagents. Cells were initially washed with cold phosphate-buffered saline (PBS) and harvested by scraping. The cells were pelleted in 1 ml of PBS at 6,000 rpm for 5 min. The pellets were washed twice with PBS and resuspended in lysis buffer (10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [Hepes], pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 25% vol/vol glycerol, 1 mM dithiothreitol [DTT], and 0.1 mM phenylmethylsulfonyl fluoride [PMSF]). The suspension was incubated for 5 min on ice and subsequently centrifuged at 6,000 rpm at 4°C for 5 min. The supernatant was discarded and one cell pellet volume of extraction buffer (20 mM Hepes, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 25% glycerol, 1 mM DTT, and 0.5 mM PMSF) was added. The suspension was incubated on ice for 15 min and then centrifuged at 14,000 rpm at 4°C for 15 min to pellet the nuclear debris. Protein concentrations of the resulting supernatants were determined using the Bradford assay (BioRad, Hercules, CA) and stored at 70°C until used for electromobility shift assays (EMSAs) or Western blot analyses.

Electromobility Shift Assay

The NF-B oligonucleotide probe used for EMSA (5'-GTGGAATTTCCTCTGA-3') corresponds to the NF-B site in the IL-8 promoter and was synthesized at the University of Cincinnati DNA Core Facility (Cincinnati, OH) (20). The probe was labeled with -[³²P]adenosine triphosphate using T4 polynucleotide kinase (GIBCO BRL) and purified in Bio-Spin chromatography columns (BioRad).

For EMSA, 10 µg of nuclear proteins were preincubated with EMSA buffer (12 mM Hepes, pH 7.9, 4 mM Tris-HCl, pH 7.9, 25 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 50 ng/ml poly [d(I-C)], 12% glycerol vol/vol, and 0.2 mM PMSF) on ice for 10 min before addition of the radiolabeled oligonucleotide probe for an additional 10 min. Protein-nucleic acid complexes were resolved using a nondenaturing polyacrylamide gel consisting of 5% acrylamide (29:1 ratio of acrylamide:bisacrylamide) and run in 0.5× TBE (45 mM Tris-HCl, 45 mM boric acid, 1 mM EDTA) for 1 h at constant current (30 mA). Gels were transferred to Whatman 3M paper, dried under a vacuum at 80°C for 1 h, and exposed to photographic film at 70°C with an intensifying screen.

Western Blot Analysis

Treated cells were washed once in PBS and lysed in ice-cold lysis buffer (50 mM Tris, pH 8.0, 110 mM NaCl, 5 mM EDTA, and 1% Triton X-100). Protein concentrations were measured using the Bradford assay (BioRad). Cytoplasmic lysates containing 50 µg of protein, or nuclear protein extracts containing 20 µg of protein, were boiled with equal volumes of treatment buffer (0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, and 10% 2--mercaptoethanol) and loaded onto 8 to 16% Tris-glycine gels (Novex, San Diego, CA). Proteins were separated electrophoretically and transferred to nitrocellulose membranes (Novex) using the Novex Xcell Mini-Gel system. For immunoblotting, membranes were blocked with 10% nonfat dried milk in Tris-buffered saline (TBS) for 1 h. Primary antibodies against either IB or the p65 subunit of NF-B (polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) were applied at 1:200 dilution for 1 h. After washing two times in TBS containing 0.05% Tween 20 (TTBS), secondary antibody (peroxidase-conjugated goat antirabbit immunoglobulin G; Calbiochem, La Jolla, CA) was applied at 1:10,000 dilution for 1 h. Blots were washed in TTBS two times over 30 min, incubated in commercial enhanced chemiluminescence reagents (Amersham, Buckinghamshire, England), and exposed to photographic film.

Treatment of Nuclear Extracts with Geldanamycin

In one group of experiments, geldanamycin was added directly to the nuclear extracts of TNF--treated cells. Cells were treated with TNF- for 1 h and nuclear proteins were extracted as described previously. The nuclear extracts of TNF--treated cells were then incubated with geldanamycin (1 µg/ml) or 0.1% DMSO for 0.5 h. After this incubation period, EMSA was performed for NF-B as described previously.

Cell Viability

Cell viability after 24 h continuous exposure to geldanamycin was measured by measuring the remaining mass of attached cells as previously described (23). Briefly, cells were washed once in PBS to remove dead/floating cells. The mass of remaining attached cells was then measured by lysing in 0.5 M NaOH and measuring DNA concentration by spectrophotometry. Percent cell viability was calculated as absorbance of treated cells/absorbance of control cells × 100.

Statistical Analysis

Differences in immunoreactive IL-8 levels, luciferase activity, and cell viability between the experimental groups were evaluated by one-way analysis of variance and Student-Newman-Keuls test. P < 0.05 was considered statistically significant.

	Results

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Geldanamycin Inhibits TNF--Mediated Production of Immunoreactive IL-8

In these experiments, we determined the effect of geldanamycin on TNF--mediated production of immunoreactive IL-8. Treatment with TNF- alone increased production of immunoreactive IL-8 compared with control cells treated with medium alone (Figure 1). Pretreatment with geldanamycin significantly inhibited TNF--mediated production of immunoreactive IL-8. Cell viability was greater than 90% after exposure to all experimental conditions (data not shown).

View larger version (8K): [in this window] [in a new window]   Figure 1.   ELISA results demonstrating the effects of geldanamycin on TNF--mediated production of immunoreactive IL-8. Control cells were maintained in basal growth medium. TNF-- treated cells were treated with TNF- (2 ng/ml) for 24 h. Geldanamycin-treated cells were treated with geldanamycin (1 µg/ml) for 1 h before the addition of TNF- for 24 h. Data represent the mean ± SEM of three separate experiments with each condition carried out in triplicate. *P < 0.05 versus TNF- alone.

Geldanamycin Inhibits TNF--Mediated IL-8 Messenger RNA Expression

Having demonstrated that geldanamycin inhibited TNF-- mediated production of immunoreactive IL-8, we next determined the effect of geldanamycin on TNF--mediated expression of IL-8 messenger RNA (mRNA). Treatment with TNF- alone increased IL-8 mRNA expression in a time-dependent manner compared with control cells treated with medium alone (Figure 2, lanes 1, 2, 4, 6, and 8). Pretreatment with geldanamycin inhibited TNF--mediated IL-8 mRNA expression at all timepoints tested (Figure 2, lanes 3, 5, 7, and 9). Collectively, these data and the previous data involving immunoreactive IL-8 demonstrate that geldanamycin is a potent inhibitor of IL-8 gene expression.

View larger version (27K): [in this window] [in a new window]   Figure 2.   Representative Northern blot (top panel) demonstrating the effect of geldanamycin on TNF--mediated expression of IL-8 mRNA. Control cells (lane 1) were maintained in basal growth medium. TNF--treated cells (lanes 2, 4, 6, and 8) were treated with TNF- (2 ng/ml) for the indicated times. Geldanamycin-treated cells (lanes 3, 5, 7, and 9) were treated with geldanamycin (1 µg/ml) for 1 h before the addition of TNF- for the indicated times. The blot is representative of three separate experiments with similar results. The bottom panel represents ethidium bromide staining of 18s ribosomal RNA (rRNA) to document equal loading of samples.

Geldanamycin Inhibits TNF--Mediated Activation of the IL-8 Promoter

Previous data involving A549 cells demonstrated that IL-8 gene expression is regulated at the transcriptional level (20, 24). Accordingly, we determined the effect of geldanamycin on TNF--mediated activation of the IL-8 promoter. Cells were transiently transfected with an IL-8 promoter- luciferase reporter plasmid and exposed to the experimental conditions. Treatment with TNF- alone induced luciferase activity ~ fivefold above control cells that were transfected and treated with medium alone (Figure 3). Pretreatment with geldanamycin significantly inhibited TNF-- mediated induction of luciferase activity. These data demonstrate that geldanamycin inhibits TNF--mediated activation of the IL-8 promoter.

View larger version (8K): [in this window] [in a new window]   Figure 3.   Luciferase assay demonstrating the effect of geldanamycin on TNF--mediated activation of the IL-8 promoter. Cells were transiently transfected with an IL-8 promoter-luciferase promoter plasmid. Control cells were maintained in basal growth medium. TNF--treated cells were treated with TNF- (2 ng/ml) for 4 h. Geldanamycin-treated cells were treated with geldanamycin (1 µg/ml) for 1 h before the addition of TNF- for 4 h. Data are expressed as fold induction over control cells and are corrected for total cellular protein. Data represent the mean ± SEM of five separate experiments with each condition carried out in duplicate. *P < 0.05 versus TNF- alone.

Geldanamycin Inhibits TNF--Mediated Activation of NF-B

Because NF-B is centrally involved in the regulation of the IL-8 promoter, we next determined the effect of geldanamycin on TNF--mediated activation of NF-B using EMSA. Treatment with TNF- alone increased activation of NF-B compared with control cells (Figure 4, lanes 1-3). The specificity of this NF-B band was demonstrated in a previous study from our laboratory involving A549 cells (25). Pretreatment with geldanamycin decreased formation of the NF-B/DNA complex (Figure 4, lanes 5 and 6), suggesting that geldanamycin inhibits activation of NF-B.

View larger version (65K): [in this window] [in a new window]   Figure 4.   EMSA demonstrating the effect of geldanamycin on TNF--mediated activation of NF-B. Control cells (lane 1) were maintained in basal growth medium. TNF--treated cells (lanes 2 and 3) were treated with TNF- (2 ng/ml) for 1 h. Geldanamycin-treated cells (lanes 4 and 5) were treated with geldanamycin (1 µg/ml) for 1 h before the addition of TNF- for 1 h. Gel represents one of three experiments with similar results.

To confirm the inhibitory effect of geldanamycin on NF-B activation, A549 cells were transiently transfected with a promoter-reporter plasmid in which three tandem NF-B sites drive luciferase expression (3×NF-B-Luc). Treatment with TNF- alone induced an ~ fourfold increase in luciferase activity above control cells transfected and treated with medium alone (Figure 5). Pretreatment with geldanamycin inhibited TNF--mediated induction of luciferase activity. Collectively, these data demonstrate that geldanamycin inhibits TNF--mediated NF-B activation in A549 cells.

View larger version (9K): [in this window] [in a new window]   Figure 5.   Luciferase assay demonstrating the effect of geldanamycin on TNF--mediated activation of NF-B. Cells were transiently transfected with luciferase reporter plasmid containing three tandem NF-B motifs. Control cells were maintained in basal growth medium. TNF--treated cells were treated with TNF- (2 ng/ml) for 4 h. Geldanamycin-treated cells were treated with geldanamycin (1 µg/ml) for 1 h before the addition of TNF- for 4 h. Data are expressed as fold induction over control cells and are corrected for total cellular protein. Data represent the mean ± SEM of five separate experiments with each condition carried out in duplicate. *P < 0.05 versus TNF- alone.

Effect of Geldanamycin on TNF--Mediated Degradation of IB

Having demonstrated that geldanamycin inhibits activation of NF-B, we next determined the effect of geldanamycin on TNF--mediated degradation of the NF-B inhibitory protein IB. Treatment with TNF- alone caused a rapid degradation of IB within 15 to 30 min, with a return to baseline levels by 60 min (Figure 6, lanes 1-4). Pretreatment with geldanamycin did not affect TNF--mediated degradation of IB (Figure 6, lanes 5-7) compared with cells treated with TNF- alone. These data suggest that the inhibitory effect of geldanamycin on NF-B activation does not involve inhibition of IB degradation in A549 cells.

View larger version (21K): [in this window] [in a new window]   Figure 6.   Representative Western blot analysis demonstrating the effect of geldanamycin on TNF--mediated degradation of IB. Control cells (lane 1) were maintained in basal growth medium. TNF--treated cells (lanes 2-4) were treated with TNF- (2 ng/ml) for the indicated times. Geldanamycin-treated cells (lanes 5-7) were treated with geldanamycin (1 µg/ml) for 1 h before the addition of TNF- for the indicated times. Gel represents one of three experiments with similar results.

Effect of Geldanamycin on Nuclear Translocation of p65 and NF-B Binding

The EMSA and luciferase reporter data provide convincing evidence that geldanamycin inhibits activation of NF-B. Whereas NF-B inhibition is usually linked to preservation of IB (12, 13), our Western blot data demonstrated that geldanamycin did not inhibit IB degradation. These findings suggest that geldanamycin may inhibit NF-B activation by preventing NF-B nuclear translocation via a non-IB-dependent mechanism. Alternatively, the findings could suggest that geldanamycin directly alters the ability of NF-B to bind DNA. To assess these two possibilities, we performed two complementary experiments. First, we determined the effect of geldanamycin on nuclear translocation of the NF-B p65 subunit using Western blot analyses of nuclear proteins. Treatment with TNF- alone caused nuclear translocation of the NF-B p65 subunit compared with control cells (Figure 7). Pretreatment with geldanamycin did not significantly affect TNF--mediated nuclear translocation of the NF-B p65 subunit. In the second experiment, we added geldanamycin (1 µg/ml for 30 min) or the DMSO vehicle (0.1% for 30 min) directly to the nuclear extracts of TNF--treated cells and performed EMSA to detect binding of NF-B. When nuclear extracts from TNF--treated cells were treated with the DMSO vehicle, there was a readily detectable signal for NF-B/DNA binding (Figure 8, lane 1). In contrast, when the nuclear extracts of TNF--treated cells were treated with geldanamycin, the NF-B/DNA complex was substantially reduced (Figure 8, lane 2). Collectively, these data indicate that geldanamycin does not inhibit nuclear translocation of NF-B but directly inhibits the ability of NF-B to bind DNA.

View larger version (17K): [in this window] [in a new window]   Figure 7.   Representative Western blot analysis of nuclear proteins demonstrating the effect of geldanamycin on TNF-- mediated nuclear translocation of the NF-B p65 subunit. Control cells were maintained in basal growth medium. TNF-- treated cells were treated with TNF- (2 ng/ml) for 1 h. Geldanamycin-treated cells were treated with geldanamycin (1 µg/ml) for 1 h before the addition of TNF- for 1 h. Gel represents one of three experiments with similar results.

View larger version (44K): [in this window] [in a new window]   Figure 8.   EMSA demonstrating the effect of geldanamycin on the ability of activated NF-B to bind DNA. (Lane 1) Nuclear extracts from TNF--treated cells were incubated with 0.1% DMSO (vehicle control) for 0.5 h. (Lane 2) Nuclear extracts from TNF--treated cells were incubated with geldanamycin (1 µg/ ml) for 0.5 h. After these incubation periods, the nuclear extracts were incubated with a radiolabeled oligonucleotide corresponding to the NF-B binding site in the IL-8 promoter.

	Discussion

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We have shown that geldanamycin inhibits TNF--mediated expression of the IL-8 gene in A549 cells. Previous reports demonstrated that geldanamycin inhibited in vitro IL-8 gene expression in a variety of cells of nonpulmonary origin without providing insight as to the mechanisms of inhibition (26). Our studies indicate that the mechanism of inhibition occurs at the level of the IL-8 promoter because geldanamycin inhibited TNF--mediated luciferase activity in A549 cells transiently transfected with an IL-8 promoter-luciferase reporter plasmid. Because IL-8 is a central chemoattractant of neutrophils at sites of inflammation, these data could partially account for the anti-inflammatory properties of geldanamycin in the lung.

The molecular regulation of the IL-8 promoter is a complex process, but it is now well established that the NF-B pathway is a central component of this process (11). With this background in mind, we examined the effect of geldanamycin on activation of NF-B. We demonstrated that geldanamycin inhibits activation of NF-B using two different assays (EMSA and NF-B-dependent luciferase activity). The use of two assays provides convincing evidence that geldanamycin is an inhibitor of NF-B activation in lung cells, thus providing a broader explanation to account for the anti-inflammatory properties of geldanamycin in the lung. A previous study demonstrated that geldanamycin inhibited NF-B activation in rat spleen cells, but the mechanism of this effect was not further elucidated (29). In murine macrophages, geldanamycin inhibited lipopolysaccharide-mediated and Taxol-mediated activation of NF-B, and the mechanism of this effect was proposed to involve inhibition of heat-shock protein 90 activity (30).

Many of the NF-B inhibitors described to date inhibit degradation of IB by either inhibiting phosphorylation of IB (31, 32) or by directly inhibiting proteasome activity (33). We were therefore surprised to find that geldanamycin did not inhibit degradation of IB, while clearly inhibiting activation of NF-B. This uncoupling of the traditional NF-B/IB pathway led us to hypothesize that geldanamycin directly blocks nuclear translocation of NF-B after it is released from IB. To test this possibility, we performed Western blot analyses on nuclear extracts from treated cells. These experiments demonstrated that in the presence of geldanamycin, TNF- stimulated nuclear translocation of the NF-B p65 subunit. Thus, it appears from these data that geldanamycin does not interfere with nuclear translocation of NF-B, thereby suggesting that geldanamycin inhibits NF-B activation by somehow inhibiting its ability to bind DNA.

To test this possibility, we added geldanamycin directly to the nuclear extracts of TNF--treated cells. These experiments demonstrated that geldanamycin decreased formation of an NF-B/DNA complex, thereby strongly suggesting that geldanamycin inhibits NF-B activation by directly modifying its ability to bind DNA. Previous studies involving herbimycin A (34) and protease inhibitors (35) lend support for this assertion. These studies also provided evidence suggesting that some inhibitors of NF-B directly modify NF-B subunits, thereby inhibiting their ability to bind DNA despite nuclear translocation. An alternative hypothesis to account for the inhibitory effect of geldanamycin could involve its ability to inhibit tyrosine phosphorylation because a previous study demonstrated that the protein tyrosine phosphatase inhibitor pervanadate activated NF-B without proteolytic degradation of IB (36).

Our interest in studying the molecular mechanisms by which geldanamycin inhibits lung cellular inflammatory responses stems from the recent observation that geldanamycin confers protection in animal models of inflammation-associated ALI. Because NF-B activation and chemokine gene expression are regarded as important mechanisms in the pathophysiology of ALI, we propose that the anti-inflammatory effects of geldanamycin observed in vivo are related to the in vitro mechanisms described in the current study. The exact mechanism by which geldanamycin inhibits NF-B binding to DNA in lung cells remains to be fully elucidated. Geldanamycin has multiple effects on signal transduction pathways, including tyrosine kinase inhibition (14), inhibition of heat-shock protein 90 activity (15, 16), and induction of heat-shock proteins (18, 19). The latter effect is particularly intriguing to us because we previously demonstrated that induction of heat-shock proteins inhibited activation of NF-B in lung cells (25). Our current data demonstrate that geldanamycin can also inhibit the ability of NF-B to bind DNA, thus further adding to the complexity of geldanamycin's pharmacologic properties. How these multiple properties interact to affect cellular proinflammatory responses in the lung will need to be better understood before giving rational consideration of geldanamycin as a pharmacologic approach to attenuate ALI.