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The Proceedings of the American Thoracic Society 1:275-281 (2004)
© 2004 The American Thoracic Society

Targeted Identification of Glucocorticoid-attenuated Response Genes

In Vitro and in Vivo Models

Jeffrey B. Smith and Harvey R. Herschman

Department of Pediatrics, Department of Biological Chemistry, and Department of Molecular Pharmacology, David Geffen School of Medicine, and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California

Correspondence and requests for reprints should be addressed to Jeffrey B. Smith, M.D., Pediatrics/Neonatology, UCLA Center for the Health Sciences, B2-325A, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail: JBSmith{at}ucla.edu


   ABSTRACT
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INITIAL GARG SCREENING: IN...
 EXPRESSION AND GLUCOCORTICOID...
 SECOND GARG SCREENING: IN...
 CHARACTERISTICS OF GARG CDNAs
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Glucocorticoids attenuate the induction of numerous inflammatory mediators. We hypothesized that a targeted screening for genes with these regulatory characteristics, called glucocorticoid-attenuated response genes (GARGs), would be an efficient way to identify genes participating in glucocorticoid-sensitive inflammatory processes. An initial application of this idea, using an in vitro model, identified 12 cDNAs induced by LPS and attenuated by dexamethasone, including a new chemokine designated LIX. In vivo studies demonstrated that endotoxemia-induced lung mRNA expression of LIX, but not of two related chemokines, is markedly enhanced by adrenalectomy and attenuated by dexamethasone. This work provided the basis for an in vivo screening project that identified 36 GARG cDNAs induced in the lung during endotoxemia. The majority represent genes of unknown function, or genes not previously implicated in the pulmonary response to inflammation. Four encode previously undescribed proteins, including a chemokine, a member of a family of guanylate-binding proteins, a 2'–5' oligoadenylate synthetase–like protein, and a novel lung-inducible Neuralized-related C3HC4 RING protein (LINCR). Our results indicate that glucocorticoid-attenuated response genes are much more diverse than originally anticipated. Future studies using microarrays in this and other inflammation models may identify many additional glucocorticoid-regulated genes potentially important in inflammatory diseases.

Key Words: asthma • chronic obstructive pulmonary disease • glucocorticoid-sensitive inflammation

Although adrenal glucocorticoid hormones have a well established role in the physiologic regulation of inflammation, our knowledge of the molecular and cellular pathways influenced by endogenous or exogenous glucocorticoids is far from complete (1–3). Despite the potential for adverse effects, therapeutic use of glucocorticoid agents has proven valuable in many inflammatory diseases, including asthma. In other conditions in which inflammation is also thought to be important, including acute adult respiratory distress syndrome or bronchopulmonary dysplasia (BPD) in neonates, the use of glucocorticoids has been disappointing. In premature infants with BPD, glucocorticoids produce demonstrable short-term improvements in respiratory function. This has led to widespread use of postnatal glucocorticoids in an attempt to prevent or treat BPD in premature infants. Unfortunately, improvements in long-term outcome have been difficult to demonstrate, and serious concerns have arisen about the potential for neurodevelopmental impairment in this population. As a result, the routine use of corticosteroids for prevention of BPD in preterm infants is no longer recommended (4). Similarly, randomized clinical trials of glucocorticoids in acute adult respiratory distress syndrome have failed to show consistent benefits (5). The limited effectiveness of glucocorticoids in BPD and acute adult respiratory distress syndrome might be due, in part, to adverse effects on lung growth and repair that offset their potentially beneficial antiinflammatory effects. Such effects could also be a limiting factor for the effectiveness of glucocorticoids in asthma and chronic obstructive pulmonary disease. If we could identify the specific glucocorticoid-sensitive inflammatory pathways important in particular lung diseases, it might be possible to develop selective antiinflammatory treatments that are safer and more effective.

One approach to identifying the glucocorticoid-sensitive pathways involved in inflammatory lung diseases is to identify genes whose expression is modulated by glucocorticoids. Although other mechanisms also contribute, a major part of the antiinflammatory actions of glucocorticoids is attributed to their ability to attenuate the induction of genes encoding a variety of mediators important in inflammatory and immune responses. Glucocorticoids inhibit the induction of the inducible form of prostaglandin H synthase (cyclooxygenase-2), the inducible form of nitric oxide synthase, and numerous inflammatory cytokines and chemokines, including interleukin (IL)-1, tumor necrosis factor-{alpha}, and IL-8 (2, 3, 6).

We refer to inflammatory stimulus-induced genes whose message expression is attenuated by glucocorticoids as glucocorticoid-attenuated response genes (GARGs) (6, 7). The hypothesis underlying our initial approach was that GARG expression characteristics define a large subset of inflammation-related genes, and that identifying these genes and determining their roles in specific disease processes could reveal new targets for therapeutic intervention. As far as we were able to determine, the GARGs known at the time we initiated our studies had all been identified either via assays of their biological activity or by procedures based on screening for inducibility. In each case, glucocorticoid attenuation was investigated after the gene or its product had been identified. Our idea was that a screening procedure directly targeting the GARGs would be an efficient way to identify glucocorticoid-sensitive, inflammation-related genes likely to have important roles in inflammatory diseases.


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We first tested this idea with a cell culture model (6). We synthesized a cDNA library in {lambda} phage, using RNA from Swiss 3T3 cells stimulated with LPS. The library was then screened by differential hybridization, using triplicate filters hybridized with an "LPS" cDNA probe synthesized from RNA from 3T3 cells treated with LPS for 2 hours, an "LPS plus Dex" probe from cells treated with LPS for 2 hours in the presence of dexamethasone, and a control cDNA probe from untreated cells. (The cells used for the LPS probe were also treated with transforming growth factor-ß, but this proved to be unimportant.) We selected as GARG candidates those clones showing both induction by LPS, manifested by increased hybridization signal with the LPS probe compared with the control probe, and attenuation by glucocorticoids, manifested by reduced signal intensity with the LPS plus Dex probe compared with the LPS probe. A single screening of 15,000 phage resulted in the identification of 12 different LPS-induced, glucocorticoid-attenuated cDNAs (Table 1) (6). Seven were known murine cDNAs. These included a cytokine and three chemokines (see Table 1). The other known cDNAs were thrombospondin-1, a secreted glycoprotein with chemotactic activity for macrophages; CYR61, a secreted growth regulator and angiogenic factor; and IRG2/IFIT3, an LPS- and interferon-induced gene of unknown function. Thus, six of the seven known GARG cDNAs encoded secreted products with known functions relevant to inflammation. This observation provided reassurance that the GARG screening procedure was successfully targeting the intended population of genes.

Five of the 12 cDNAs were previously undescribed murine sequences. The high proportion of new sequences found in this small-scale screening provided support for the idea that a targeted GARG screening had the potential to identify interesting new genes. One of the five new murine sequences encodes a new chemokine, LPS-induced CXC chemokine (LIX, murine CXCL5). LIX contains the Glu-Leu-Arg (ELR) motif typical of neutrophil-chemoattractant chemokines and proved to be a potent neutrophil chemoattractant (8). LIX is described in more detail below, because it became the model gene for our second GARG screening. Two of the new cDNAs encode members of a family of LPS- and interferon-inducible genes in which we identified multiple conserved tetratricopeptide repeat domains (6, 9). The functions of these interferon-inducible tetratricopeptide repeat domain (IFIT) genes are still unknown. The other two GARG clones contained partial sequences of cDNAs later cloned and characterized by others. One encodes p62/ZIP ({zeta}-interacting protein), a ubiquitin-binding cytosolic phosphoprotein that interacts with several signal transduction molecules, including the tyrosine kinase p56lck and protein kinase C-{zeta} (10). The other encodes mafF, a regulatory component of the Maf family of transcription factors (11).


   EXPRESSION AND GLUCOCORTICOID REGULATION OF LIX
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When LIX was first identified, its relationship to other murine and human ELR+ CXC chemokines was unclear. An effort to identify a possible novel human LIX homolog resulted in our cloning the human GCP-2 (granulocyte chemotactic protein-2, CXCL6) gene as well as the previously cloned ENA-78 (epithelial cell–derived neutrophil attractant-78, CXCL5) gene (12). Comparative sequence analysis suggested that human ENA-78 and GCP-2 are the result of evolutionarily recent gene duplication (13). This suggestion was confirmed by later analysis of murine and human genome sequence data, which showed that LIX is part of a minicluster with the platelet basic protein (PBP) and platelet factor-4 (PF4) genes in the murine genome. This minicluster is represented in the human genome by duplicated loci containing ENA78–PBP–PF4 and GCP2–{psi}PBP–PF4V (14). Thus, human ENA-78 and GCP-2 are equally close homologs of murine LIX.

In the Swiss 3T3 cells used for the screening, LIX is strongly induced by LPS, and its induction is markedly attenuated (80% reduction) by dexamethasone (6). Unlike the related murine ELR+ CXC chemokines KC and macrophage inflammatory protein-2 (MIP-2), LIX is not expressed in macrophages. To further investigate the expression of characteristics of LIX and its regulation by glucocorticoids, we compared the expression of LIX, KC, and MIP-2 in vivo using an acute endotoxemia model. We found, first, that LIX, KC, and MIP-2 have distinct induction kinetics and expression patterns during endotoxemia. After intravenous administration of LPS, LIX message expression peaks later (4 hours) than expression of KC (2 hours) or MIP-2 (1 hour) and remains elevated longer (15). Although all three chemokines are induced in multiple tissues, they have distinct patterns of expression in different tissues. For example, endotoxemia-induced LIX expression is particularly abundant in the heart, with message levels five- to sixfold greater than in lung and spleen, and 20-fold greater than in liver. In contrast, endotoxemia-induced KC expression is equal in heart, lung, and liver, whereas MIP-2 expression is strongest in the lung. The observation of high LIX expression in the heart led to the finding that LIX (but not KC or MIP-2) is induced in cardiomyocytes during ischemia–reperfusion, and that neutrophil recruitment to reperfused rat myocardium is due mainly to LIX (16).

When we evaluated the effect of glucocorticoids on LIX induction in the endotoxemia model, we were initially surprised to find that pretreatment with dexamethasone fails to reduce the level of LIX message induced in the lung. These experiments were done in mice with an intact hypothalamic–pituitary–adrenal axis. Further investigation using adrenalectomized (ADX) mice showed that rather than being insensitive to glucocorticoids in vivo, expression of LIX is extremely sensitive to glucocorticoids (15). In control mice not injected with LPS, basal LIX message expression in the lung is increased in ADX mice compared with normal mice (Figures 1A and 1C), and dexamethasone suppresses the basal level of LIX expression in both groups. Injection of LPS in ADX mice induces LIX expression in the lung to a level 10-fold greater than in LPS-injected normal or sham-operated mice (Figures 1B and 1C). Dexamethasone reduces the level of LIX message in LPS-treated ADX mice down to the same level as in LPS-treated normal or sham-operated mice with or without dexamethasone. We conclude that dexamethasone has no effect in normal mice because LPS-induced LIX expression in the lung is already maximally attenuated by the endogenous glucocorticoid response to endotoxemia. The magnitude of the enhancement of LPS-induced LIX expression in ADX mice is tissue specific, with the largest effects seen in lung (10-fold) and small intestine (24-fold). In contrast to the expression of LIX, endotoxemia-induced lung expression of KC or MIP-2 is not enhanced by adrenalectomy and is not attenuated by dexamethasone in ADX mice (15). These observations emphasize the importance of in vivo studies for understanding glucocorticoid effects on lung inflammation and helped define the model system we used for further GARG screening studies.



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  		Figure 1. Glucocorticoid regulation of basal and endotoxemia-induced LIX expression in the lung. Normal (Norm), adrenalectomized (ADX), and sham-adrenalectomized (Sham) mice were treated with two doses of dexamethasone (DEX, 400 µg) or with two doses of saline 16 to 20 hours before and immediately before tail vein injections of 50 µg of LPS or saline. Lung RNA harvested 4 hours after LPS or saline injection was analyzed by Northern blotting. The ribosomal protein S2 message was used as a control for variations in loading. (A and B) Representative blots illustrating ADX-induced increases in basal and endotoxemia-induced LIX expression, respectively. Film exposure times for the LIX panels were 9 days (A) and 24 hours (B). (C) LIX message expression (mean ± SEM, three mice per group). The scale is set so that LIX expression in the LPS-treated sham-operated mice (without dexamethasone) equals 1.0. *p < 0.05 compared with LPS-treated normal and sham-operated mice; {maltese cross}p < 0.05 compared with basal expression in normal and sham-operated mice. Reprinted by permission from Rovai and coworkers (15).

 

   SECOND GARG SCREENING: IN VIVO MODEL
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On the basis of the studies of LIX described above, we undertook a screening designed to identify endotoxemia-induced, glucocorticoid-attenuated genes expressed in the lung. Adrenalectomized mice were used for the screening to eliminate the effects of endogenous glucocorticoids and to take advantage of potential increases in message induction and glucocorticoid attenuation of candidate genes, as seen for LIX (Figure 1). Because LPS initiates the release of endogenous mediators, including tumor necrosis factor-{alpha}, IL-1ß, and IFN-{gamma}, we expected to clone genes responsive to these and additional downstream mediators, as well as genes directly responsive to LPS. Although the timing and pattern of gene expression would surely differ in other models, we postulated that GARGs identified with the endotoxemia model would be good candidates for further investigation in other inflammatory disease models, both acute and chronic.

When this work was initiated, high-density microarrays were not yet available. We used the suppression–subtraction hybridization technique (17) to construct a subtracted library enriched in endotoxemia-induced genes from lung RNA of ADX mice, and then screened the subtracted library by differential hybridization to select candidate GARG clones (18). The "tester" cDNA (containing the differentially expressed cDNA population to be enriched by the subtraction) was prepared from lungs of LPS-treated ADX mice. The "driver" cDNA (used for subtracting nondifferentially expressed cDNAs from the tester population) was prepared from lungs of dexamethasone-treated ADX mice. The differential hybridization screening was performed by hybridizing replicate filter lifts of the subtracted library phage with (1) an "LPS" cDNA probe synthesized from lung RNA of ADX mice killed 2 hours after intravenous injection of LPS, (2) an "LPS plus Dex" probe from lung RNA of ADX mice pretreated with dexamethasone and killed 2 hours after intravenous injection of LPS, and (3) a control cDNA probe from lung RNA of ADX mice treated with dexamethasone. The mice received a single 50-µg dose of LPS (or saline) intravenously. Two 400-µg doses of dexamethasone (or vehicle) were administered subcutaneously 18 hours before and 5 minutes before LPS. As in the in vitro screening, phage plaques selected as candidates were required to show both induction in response to endotoxemia and attenuation of induction by dexamethasone. More than 90% of the library clones screened satisfied the first criterion, verifying that the library was highly enriched in endotoxemia-induced genes. A screening of 6,600 plaques yielded 599 candidate GARG phage satisfying both criteria (18).

After elimination of replicate phage via a sequential hybridization procedure (18), the expression characteristics of the remaining 55 distinct candidates were evaluated by Northern blotting. First, induction during endotoxemia and attenuation by dexamethasone of all candidates were evaluated in normal (nonoperated) mice. Our criteria for a GARG message were an endotoxemia-induced increase in lung message expression of twofold or more and attenuation by dexamethasone of 25% or more of the endotoxemia-induced increase. Thirty-five of the 55 candidate cDNAs met both of these criteria in normal mice. Next, we evaluated the effect of adrenalectomy on the expression characteristics of the remaining 20 candidates, and on selected genes already confirmed as GARGs in normal mice. We hoped that the absence of endogenous glucocorticoids in ADX mice would reveal attenuation by dexamethasone for a number of remaining candidates, as observed for LIX (Figure 1). However, only one additional candidate, P-selectin, was identified as glucocorticoid attenuated in ADX mice but not in normal mice, bringing the total of confirmed GARGs isolated in the screening to 36. This suggested that genes with expression characteristics like those of LIX and P-selectin (maximally attenuated by the endogenous glucocorticoid response in this model) constitute only a small proportion of the GARG population.

The 36 GARG cDNAs identified in the screening are listed in Table 2. Four additional genes previously identified as GARGs (IFN-{gamma}, IL-6, LIX, and the monokine induced by interferon-{gamma} [MIG]) are included in Table 2 for comparison. Although all the genes in Table 2 are induced by LPS and attenuated by dexamethasone, they exhibit wide quantitative differences in responses to LPS and dexamethasone compared with normal mice (18). They also exhibit both quantitative and qualitative differences in the effect of adrenalectomy (Figure 2). Adrenalectomy produces marked enhancement of LPS-induced expression of LIX (10-fold) and IL-6 (eightfold), and twofold or greater enhancement of LPS-induced expression of intercellular cell adhesion molecule-1 (ICAM-1), P-selectin, IL-1ß, MCP-1, MIP-1{alpha}, and interferon-inducible GTPase (IIGP). In contrast, adrenalectomy reduces the LPS-induced expression of the chemokines MIG and interferon-inducible T cell {alpha}-chemoattractant (I-TAC) by 40 to 50%. (I-TAC is described in more detail below.) The mechanisms responsible for these gene-specific effects of adrenalectomy remain to be elucidated.



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  		Figure 2. Lung message expression of representative GARG cDNAs. Lung RNA from normal, adrenalectomized (ADX), and sham-adrenalectomized (Sham) mice was collected 4 hours after LPS injection, with or without dexamethasone (DEX) pretreatment (five mice per group), as in Figure 1. Replicate Northern blots of pooled RNA specimens were hybridized with probes for GARG candidates and S2. CIG, cytomegalovirus-induced IFN-{gamma} responsive; GBP = guanylate-binding protein; IGTP = interferon-inducible guanosine triphosphate (GTP)–binding protein; IRF, interferon regulatory factor; I-TAC = IFN-inducible T cell {alpha}-chemoattractant; LINCR = lung-inducible Neuralized-related C3HC4 RING protein; LIX = LPS-induced CXC chemokine; MCP = monocyte chemoattractant protein; MIG = monokine induced by IFN-{gamma}; OASL = oligoadenylate synthetase–like. Reprinted by permission from Smith and coworkers (18).

 

   CHARACTERISTICS OF GARG CDNAs
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As expected, some of the cDNAs identified in the in vivo screening encode well known proinflammatory mediators, including inflammatory cytokines, chemokines, and adhesion proteins (Table 2). The presence of suppressor of cytokine signaling-3 (SOCS-3) (19) serves as an important reminder that antiinflammatory as well as proinflammatory mediators may have GARG expression characteristics. In addition, many of the GARG cDNAs represent genes of unknown function, or genes not previously associated with lung inflammation. All are candidates for further study to determine their functional roles in a variety of lung disease models, including those relevant to asthma and chronic obstructive pulmonary disease. The collection as a whole should also be useful in studies of the molecular mechanisms by which glucocorticoids attenuate gene expression (2, 3).

The GARG cDNAs include members of diverse functional and structural categories of genes. About half of the cDNAs were originally identified as interferon-inducible genes. These include the chemokines IP-10 and MIG (20), the three members of the IFIT family cloned in our earlier GARG screening (6, 9), and multiple members of the guanylate-binding protein (GBP) and IRG-47 families of interferon-inducible guanosine triphosphate (GTP)–binding proteins (IGTPs) (21–26).

The diversity of the GARGs is exemplified by the four new murine cDNAs identified in this study. One encodes the murine ortholog of a human chemokine designated I-TAC (CXCL11) (27). I-TAC is closely related to IP-10 and MIG. Murine I-TAC, like its human counterpart, binds and activates the receptor CXCR3, which is also the receptor for IP-10 and MIG, and is expressed primarily on activated Type 1 helper T lymphocytes and natural killer cells in both species (28).

The second new cDNA encodes a new member of the GBP family, murine GBP-5. We also identified an alternatively spliced form that lacks part of the GTP-binding motif and differs at the C terminus, the first example of alternate splicing described for this gene family (29). The GBPs are large GTPases distinguished by their ability to bind with equal affinity to GTP, guanosine diphosphate (GDP), and guanosine monophosphate (GMP), and by their ability to catalyze the hydrolysis of GTP to GMP as well as GDP (30, 31). Studies indicate that GBP family members influence cell proliferation and have antiviral effects (25, 26, 32–34).

The third new cDNA encodes a new member of a small family of oligoadenylate synthetase–like (OASL) genes (18). The N-terminal portion of the OASL proteins consists of an approximately 340-residue domain. The 2'–5' OAS enzymes control a regulated RNA decay pathway involved in the antiviral and growth inhibitory effects of interferons (35). In addition, the OASL proteins have a C-terminal region containing two tandem ubiquitin-like domains not present in the OAS proteins (36). Although the functions of the OASL proteins are unknown, a report indicates that the ubiquitin-like domain of one OASL family member specifically interacts with the transcriptional repressor methyl CpG-binding protein-1 (37), suggesting a possible role for OASL proteins in transcriptional regulation.

The fourth new cDNA encodes a novel lung-inducible Neuralized-related C3HC4 RING domain protein (LINCR) not previously described in any species (18). As with LIX, basal expression of LINCR is enhanced in ADX mice, but unlike in the case of LIX, endotoxemia-induced LINCR expression is not affected by adrenalectomy (Figure 3A). On the basis of the murine sequence, we identified the human LINCR homolog by analysis of genome sequence data. The murine LINCR cDNA encodes a 254-amino acid residue protein related to, but distinct from, the mammalian homologs of Drosophila Neuralized (Neur). Drosophila Neur and its homologs in other species contain two copies of a distinctive 153- to 156-amino acid region known as the Neur repeat (NR) domain (Figure 3B). In contrast, murine and human LINCRs contain only a single NR domain. Phylogenetic analysis of the NR domains suggests that the ancestral mammalian LINCR gene may have evolved from a duplicated Neur gene by deletion of its second NR domain. The LINCR and Neur proteins all contain C3HC4-type RING domains of similar structure near their carboxy termini. The RING domain is a key feature of a major class of ubiquitin E3 ligase enzymes that are responsible for substrate-specific transfer of ubiquitin to target proteins (38). Drosophila and Xenopus Neur are ubiquitin E3 ligases that influence cell fate decisions by targeting Delta, a ligand of the developmentally important Notch receptor (39–42). Mammals have four Notch receptors and five Notch ligands homologous to those in Drosophila. Accumulating evidence indicates that the Notch system has important roles in both embryonic development and tissue homeostasis in a wide variety of mammalian organ systems, including the lung (43–48). We have shown that LINCR is a functional E3 ligase (T. T. Nguyen and J. B. Smith, unpublished results). On the basis of the structural and enzymatic similarity of LINCR to Neur, we speculate that LINCR may, like Neur, modulate Notch signaling. If so, LINCR could play an important role in the network of interactions linking inflammation with pathways important in lung development and repair.



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  		Figure 3. Glucocorticoid regulation of LINCR, and structures of LINCR and Neuralized. (A) LINCR message expression (mean ± SEM) in the lungs of normal, adrenalectomized (ADX), and sham-adrenalectomized (Sham) mice treated with LPS ± dexamethasone (DEX), as in Figures 1 and 2. Basal LINCR message expression was enhanced in ADX mice, but adrenalectomy did not enhance LINCR induction by LPS. (B) Structure of LINCR and Neuralized (Neur) proteins, showing the Neur repeat (NR) and RING (R) domains. C.e. = Caenorhabditis elegans. Adapted by permission from Smith and coworkers (18).

 
CONCLUSIONS
Before this work was done, we and others expected that the glucocorticoid-attenuated subset of inflammation-induced genes would be found to encode mainly mediators such as cytokines, chemokines, receptors, adhesion molecules, or enzymes such as cyclooxygenase-2 and the inducible form of nitric oxide synthase. In fact, genes of these types represent only about one-third of the lung-expressed GARGs (Table 1). The results of our screening suggest that the network of glucocorticoid-attenuated genes participating in the lung response to endotoxemia is much more diverse than previously thought. Moreover, the responses to glucocorticoids and to adrenalectomy differed qualitatively and quantitatively among the GARG cDNAs, suggesting that mechanisms of glucocorticoid regulation differ among the GARGs.

To the best of our knowledge, the screenings described here (6, 18) are the only studies reported to date that specifically target the identification of glucocorticoid-attenuated response genes in any inflammatory model. We suggest that this approach can be fruitfully applied to a wide variety of both acute and chronic inflammatory disease models. Microarray and proteomic techniques would likely be the methods of choice for future studies. We expect that different (but overlapping) sets of genes will be identified in other inflammatory models and other tissues. The 40 cDNAs listed in Table 2 probably represent only a fraction of the GARGs involved in the lung response to endotoxemia. We know that the differential screening was not exhaustive, because many of the cDNAs were single isolates, and because several known lung-expressed GARGs, including IFN-{gamma}, IL-6, LIX, and MIG, were not identified in the screening. Injection of LPS is expected to trigger a cascade of gene induction, so we would expect to identify additional genes if the evaluation were performed at a range of time points. Taken together, these observations suggest that developing a comprehensive understanding of the actions of glucocorticoids on the gene networks involved in inflammation-related processes will be a tremendous challenge. We are just beginning to appreciate the full complexity of the mechanisms involved in the antiinflammatory actions of glucocorticoids.


   ACKNOWLEDGMENTS
 
J.B.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.R.H. has owned Merck, Pharmacia, and Pfizer stock for more than 3 years.


   FOOTNOTES
 
Supported by National Institutes of Health grants HL57008 (J.B.S.) and GM24797 (H.R.H.), and by contract DE FC0387ER60615 between the Regents of the University of California and the Department of Energy.

(Received in original form February 18, 2004; accepted in final form April 20, 2004)


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