INTRODUCTION

TOP ABSTRACT INTRODUCTION MOLECULAR BIOLOGY OF NF-B BACTERIA-INDUCED NF-B... ROLE OF NF-B IN... MODULATION OF NF-B ACTIVATION... INTERACTIONS BETWEEN NF-B AND... SUMMARY REFERENCES

THE LUNG IS AN IMPORTANT TARGET ORGAN for systemic inflammatory mediators released after major trauma (6, 39, 44) and severe infection (113, 118). Acute respiratory distress syndrome (ARDS), clinically manifested as inflammatory lung injury of very rapid onset, is characterized by intractable respiratory dysfunction. This syndrome is associated with hypoxemia, reduced lung compliance, and diffuse infiltrations (13). Neutrophils sequestered in the lung play a central role in the pathogenesis of ARDS (50). Lung neutrophil accumulation seen in ARDS is thought to occur as a result of a cascade of cellular events initiated by either infectious or noninfectious inflammatory stimuli. Local activation of resident cells in the lung interstitium and alveolus, primarily macrophages, leads to elaboration of several proinflammatory cytokines such as tumor necrosis factor (TNF)- and interleukin (IL)-1 and chemokines including IL-8 and macrophage inflammatory protein (MIP)-2 (35, 37, 47, 87). These mediators act in concert to promote neutrophil sequestration by activating endothelial cell adhesion molecule expression and to induce migration of neutrophils into the interstitium and alveolar space where they propagate inflammation and injury through the release of reactive oxygen species (ROS) and proteolytic enzymes (155). A counterinflammatory response in the lungs is also initiated with the release of anti-inflammatory cytokines such as IL-10, IL-4, IL-13, IL-1 receptor antagonist (IL-1RA), and transforming growth factor (TGF)- (36, 47, 109). Elevated levels of proinflammatory mediators combined with decreased expression of anti-inflammatory molecules correlate with the magnitude of lung injury and its outcome (108). The imbalance between proinflammatory and anti-inflammatory mechanisms is a critical component of lung inflammation.

The signals that lead to increased gene expression and biosynthesis of proinflammatory mediators by airspace inflammatory and epithelial cells are thus of considerable interest. An understanding of the expression of genes, in particular the transcriptional apparatus usually located in the upstream region of the gene promoter, is required to explain the regulation of expression of proinflammatory genes. Nuclear factor (NF)-B is one such critical transcription factor required for maximal expression of many cytokines involved in the pathogenesis of ARDS. NF-B, first identified by Sen and Baltimore (124), functions to enhance the transcription of a variety of genes, including cytokines and growth factors, adhesion molecules, immunoreceptors, and acute-phase proteins (a list of genes containing NF-B sites in their promoters is shown in Table 1). Under resting condition, NF-B functions in regulating the expression of genes involved in normal immunologic responses such as the generation of antibody light chains and other immunoregulatory molecules (124, 152). NF-B activation is necessary for an intact host defense response, whereas excessive activation of NF-B results in overly exuberant inflammatory injury of lungs and other organs (17, 18, 40).

View this table: [in this window] [in a new window]   Table 1.   Lung injury relevant genes regulated by NF-B

In this review, we focus on the recent progress in our understanding of the role of NF-B and interactions between NF-B and other transcription factors in the pathogenesis of acute lung injury.

	MOLECULAR BIOLOGY OF NF-B

TOP ABSTRACT INTRODUCTION MOLECULAR BIOLOGY OF NF-B BACTERIA-INDUCED NF-B... ROLE OF NF-B IN... MODULATION OF NF-B ACTIVATION... INTERACTIONS BETWEEN NF-B AND... SUMMARY REFERENCES

The NF-B family of transcription factors. NF-B belongs to the Rel family of proteins that are ubiquitous transcription factors sharing a common structural motif for DNA binding and dimerization (10). Although, for the sake of simplicity, NF-B is often referred to as a single entity, it is, in reality, a complex mixture of homodimers and heterodimers, all with distinct characteristics and biological properties. The cloning of NF-B family members has revealed five distinct subunits homologous to the protooncogene c-Rel and the Drosophila melanogaster morphogen dorsal: NF-B1 (p50/105), NF-B2 (p52/100), Rel A (p65), Rel B, and c-Rel itself (10, 134). These subunits are all highly related over an ~300-residue amino-terminal DNA binding and dimerization domain termed the Rel homology domain.

Activation of NF-B. NF-B is normally sequestered in the cytoplasm through its association with an inhibitor protein called inhibitory B (IB), which masks the nuclear translocation signal and thus prevents NF-B from entering the nucleus. NF-B activation represents the terminal step in a signal transduction pathway leading from the cell surface to the nucleus. On exposure of the cell to activation signals such as the binding of lipopolysaccharide (LPS), TNF-, or IL-1 to cell surface receptors, the IB protein is phosphorylated on serine-32 and serine-36, ubiquinated, and degraded in proteasomes. After being freed from association with IB, the NF-B complex moves to the nucleus where it binds to specific sequences in the promoter/enhancer regions of genes.

NF-B inhibitors and their regulation. The various forms of IB include IB-, IB-, IB-, and Bcl-3 (10, 45, 153). In addition, p105, which is the precursor of p50, and p100, which is the precursor of p52, can bind Rel A and thus function as NF-B inhibitors (31, 83, 97). The COOH-terminal portions of p105 and p100 have been designated IB- and IB-, respectively. These inhibitory units contain multiple copies of ankyrin repeat domains that allow interaction with NF-B in a configuration that masks the nuclear localization signal domains of NF-B, thereby preventing nuclear transport (7). Signaling pathways leading to NF-B activation converge on phosphorylation, ubiquitination, and degradation of IBs (except Bcl-3), which unmask the nuclear localization signal leading to translocation of NF-B/Rel dimers into the nucleus (4, 86, 125). IBs are also active in the nucleus. IB- and IB- interact with p50/p65 NF-B heterodimers in the nucleus as in the cytoplasm, inhibiting transcriptional activity of NF-B (4, 144). In experiments examining NF-B activation in the lung, levels of IB- in the nucleus as well as in the cytoplasm were elevated in a mixed population of intraparenchymal lung cells after hemorrhagic shock in association with NF-B nuclear translocation (94). These findings suggest that 1) IB- is upregulated by NF-B and 2) other IB proteins contribute to NF-B activation in the lung after blood loss.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Pathways of inhibitory B (IB) regulation showing the upstream components signaling phosphorylation of IB and the release of nuclear factor (NF)-B. TRAF, tumor necrosis factor (TNF) receptor-associated factor; NIK, NF-B-inducing kinase; MEKK, mitogen-activated protein kinase kinase kinase; IKK, IB kinase; IKAP, IKK-associated protein; P, phosphorylation.

	BACTERIA-INDUCED NF-B ACTIVATION: ROLE OF TOLL-LIKE RECEPTORS

TOP ABSTRACT INTRODUCTION MOLECULAR BIOLOGY OF NF-B BACTERIA-INDUCED NF-B... ROLE OF NF-B IN... MODULATION OF NF-B ACTIVATION... INTERACTIONS BETWEEN NF-B AND... SUMMARY REFERENCES

Infection with microbial pathogens and septicemia are major causes of acute lung injury. Major causative agents include gram-negative and/or gram-positive bacteria. Septic shock and acute lung injury follow a pattern in which one or more products of microbial pathogens such as the endotoxin of gram-negative bacteria, diverse membrane components of gram-positive bacteria, and nonmethylated CpG-rich sequences of bacterial DNA activate the innate immune system. This system represents the immediate host defense mechanism that involves the secretion of cytokines and other mediators with antimicrobial effects. It is well documented that phylogenetically conserved signaling mechanisms provide an immediate cellular reaction that utilizes NF-B at the center of its first line of defense (89). The recognition of a pathogen is mediated by a set of germ line-encoded receptors referred to as pattern-recognition receptors, which directly recognize invariant molecular structures (pathogen-associated molecular patterns). These are shared by large groups of microorganisms (56, 88). The three functional classes of pattern-recognition receptors are signaling receptors, endocytic receptors, and secreted proteins. Studies over the past few years have demonstrated that a family of signaling receptors, known as the Toll-like receptors (TLRs), plays a crucial role in Drosophila and mammalian host defense. In both organisms, TLRs activate intracellular signaling, notably via NF-B. At least nine TLRs have been identified in humans, but only three of them have been characterized as interacting with a ligand of bacterial origin (89, 116, 142). TLR2 is a receptor for peptidoglycan and lipopeptide from gram-positive bacteria and zymosan from yeast. TLR4 recognizes LPS from gram-negative bacteria (reviewed in Ref. 102). It was recently shown that TLR9 is a putative receptor for bacterially derived CpG DNA (51). All members of the Toll family are membrane proteins that span the membrane once and share similar extracellular domains, which include 18-31 leucine-rich repeats and similar cytoplasmic domains of ~200 amino acids. Interestingly, the latter domain is also similar to the cytoplasmic domain of the IL-1 receptor [Toll/IL-1 receptor homologous region (TIR)]. The cytoplasmic domains of the Toll family define a subclass of TIR domains. The TIR domain of human TLR4, for example, is 32% identical to Drosophila Toll but less similar (20% identical) to the TIR domain of the IL-1 receptor.

Specific components of microbial cell walls are strong activators of innate immune responses. Human pathogen-associated molecular patterns such as LPS of gram-negative bacteria and peptidoglycan and lipoteichoic acid components of gram-positive bacteria trigger the production of IL-1, TNF- (103, 122, 141, 157), and IL-6 (74) as well as of cyclooxygenase metabolites (114). Recent genetic and biochemical experiments have highlighted the critical role of TLRs in LPS-induced NF-B activation (27, 60, 100). Importantly, the responsiveness of TLRs to LPS is dependent on LPS binding protein and CD14 (121, 139). On binding of LPS to TLRs via CD14, a number of molecules are recruited to the receptor to mediate NF-B activation (Fig. 2). First, the adaptor molecule myeloid differentiation factor 88 (MyD88) binds to the receptor and interacts through its death domain with the protein kinase IL-1 receptor-associated kinase (IRAK), which, in turn, recruits the adaptor TRAF-6 to the receptor complex (49). The importance of MyD88 in LPS signaling was confirmed by analysis of MyD88-deficient mice, which are normally highly resistant to LPS-induced shock (62). Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) bridges TRAF-6 to MEKK1. This protein is specific for the Toll/IL-1 pathway and is a regulator of MEKK1 processing (64). Expression of wild-type ECSIT accelerates the processing of MEKK1, whereas a dominant negative fragment of ECSIT blocks MEKK1 processing and activation of NF-B (64). A recent study (3) indicated that stimulation of TLR2 by Staphylococcus aureus induces NF-B activation through the small GTPases Rac1 and Cdc42.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Signaling pathway linking Toll-like receptor (TLR) to NF-B. On binding of lipopolysaccharide (LPS) to TLRs via CD14, the adaptor molecule myeloid differentiation factor 88 (MyD88) binds to the receptor and interacts through its death domain with the interleukin (IL)-1 receptor-associated kinase (IRAK), which, in turn, recruits the adaptor TRAF-6 and the evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) to the receptor complex. Downstream components MEKK1, MEKK2, and MEKK3 or NIK, which are activated by TAK1, link the receptor complex and phosphorylation of IKK- and IKK- in IKK complex. IKK complex phosphorylates IB-, IB-, and IB- at amino-terminal serines and results in degradation of the inhibitors by the ubiquitin-proteasome pathway and the subsequent release of NF-B. LBP, LPS binding protein.

Resuscitated hemorrhagic shock is believed to promote the development of lung injury by priming the immune system for an exaggerated inflammatory response to a second, often trivial, stimulus, the so-called "two-hit hypothesis" (41, 95). In recent studies, a rodent model of LPS-induced lung injury after resuscitated hemorrhagic shock was used to examine the regulation of TLR4 gene and protein expression in vivo and to evaluate the impact of its regulation on the development of inflammation (Fan J and Rotstein OD, unpublished data). The studies have demonstrated that intratracheal LPS alone induces a rapid reduction of TLR4 mRNA in the whole lung and recovered alveolar macrophages. This effect appeared to be due to a lowering of TLR4 mRNA stability by ~69%. In contrast, although shock and resuscitation alone have no effect on TLR4 mRNA levels, they markedly alter the response to LPS. Specifically, the antecedent shock prevented the LPS-induced reduction in TLR4 mRNA levels in whole lung tissue and alveolar macrophages. This reversal can be explained by the ability of prior resuscitated shock to prevent the destabilization of TLR4 mRNA by LPS as well as to augment LPS-stimulated TLR4 gene transcription compared with LPS alone. Oxidant stress related to shock or resuscitation appears to contribute to the regulation of TLR4 mRNA. Supplementation of the resuscitation fluid with the antioxidant NAC reversed the ability of shock or resuscitation to preserve TLR4 mRNA levels after LPS. TLR4 protein levels in whole lung and surface expression on alveolar macrophages mirrored the changes seen in TLR4 mRNA. Furthermore, preservation of surface TLR4 receptors in LPS-treated alveolar macrophages recovered from shock or resuscitated animals correlated with enhanced NIK activity, NF-B nuclear translocation, and inflammatory cytokine gene transcription (Fan J and Rotstein OD, personal communication). These data suggest that Tlr4 expression is controlled both transcriptionally as well as posttranscriptionally through altered mRNA stability and that changes in the local cellular microenvironment profoundly influence the net effect of these processes and, ultimately, cell responsiveness.

	ROLE OF NF-B IN THE LUNG INFLAMMATORY RESPONSE

TOP ABSTRACT INTRODUCTION MOLECULAR BIOLOGY OF NF-B BACTERIA-INDUCED NF-B... ROLE OF NF-B IN... MODULATION OF NF-B ACTIVATION... INTERACTIONS BETWEEN NF-B AND... SUMMARY REFERENCES

Increasing evidence shows an important role for NF-B in the pathogenesis of acute lung inflammation. In vitro studies have shown that NF-B regulates gene expression of cytokines (TNF-, IL-1), chemokines [MIP-2, cytokine-induced neutrophil chemoattractant (CINC)], and adhesion molecules [intercellular adhesion molecule (ICAM)-1, E-selectin] (see Ref. 17). All of these factors play an important role in lung inflammatory injury (30). These in vitro findings are supported by a study in humans with ARDS (123) showing enhanced NF-B activation in alveolar macrophages recovered by bronchoalveolar lavage. In addition, an in vivo study in animals (82) has shown an association between NF-B activation and the expression of cytokines, chemokines, and vascular adhesion molecules (82). Recent studies in vivo demonstrated that lung NF-B activation is suppressed by antioxidants (16, 76-78) or anti-inflammatory cytokines (70), resulting in decreased proinflammatory mediator expression and reduced inflammatory injury. Thus it appears that activation of NF-B is central to the development of pulmonary inflammation and acute lung injury.

Lung neutrophil activation and apoptosis. Acute lung injury is characterized by the accumulation of neutrophils in the lungs, accompanied by the development of interstitial edema and an intense inflammatory response. A study (2) has addressed the role of neutrophils as early immune effectors in hemorrhage- or endotoxemia-induced lung injury. Mice were made neutropenic with cyclophosphamide or anti-neutrophil antibodies, and it was shown that endotoxemia- or hemorrhage-induced lung edema was significantly reduced in those animals (2). Activation of NF-B after hemorrhage or endotoxemia was also diminished in the lungs of neutropenic mice compared with the lungs in nonneutropenic mice. These experiments show that neutrophils play a central role in initiating acute inflammatory responses and inducing tissue injury in lungs after hemorrhage or endotoxemia.

Alveolar macrophages. Alveolar macrophages are strategically situated at the air-tissue interface in the alveoli and alveolar ducts and are therefore the first cells encountered by inhaled organisms and antigens in the lower respiratory tract. Alveolar macrophages not only act in their traditional role as phagocytes but also function as potent secretory cells. Alveolar macrophages, on appropriate simulation, can release a wide variety of biologically active products and thereby play an important role in regulating inflammatory reactions within the lung. Depletion of alveolar macrophages by intratracheal instillation of liposomes containing the compound dichloromethylene diphosphonate has been used to study alveolar macrophage functions in vivo (23, 30, 129). It has been shown with this technique that depletion of alveolar macrophages reduces lung production of TNF- and neutrophil recruitment in a model of LPS-induced lung inflammation (12, 48). In a model of bacterial pneumonia (23), depletion of alveolar macrophages by dichloromethylene diphosphonate-liposomes unexpectedly caused increased lung production of TNF- and increased lung neutrophil recruitment. Although the nature of the stimulus may dictate the function of alveolar macrophages, it appears from the latter study that sources of TNF- other than alveolar macrophages, under special circumstances, are important to the development of lung inflammatory injury.

Endothelial activation. Endothelial expression of molecules that mediate adhesion and signaling of leukocytes is the key to understanding the transcriptional mechanisms of acute lung injury (28, 43, 79, 110). A significant advance is the concept that only the activated endothelium participates in the inflammatory response (150). In lung injury, endothelial adhesion molecules have a role in recruiting inflammatory cells such as neutrophils and lymphocytes from the circulation to the site of inflammation. NF-B regulates the expression of several genes that encode adhesion molecules such as ICAM-1, vascular cell adhesion molecule-1, and E-selectin. Cytokine-induced cell surface expression of E-selectin, vascular cell adhesion molecule-1, and ICAM-1 and the secretion of IL-8 as well as of other chemokines are regulated at the transcriptional level in endothelial cells by the binding of NF-B to its putative site in the 5'-flanking sequences (8). NF-B-dependent expression of these molecules is downregulated in endothelial cells by treatment with antioxidants (25). This finding suggests a central role for NF-B in the activation of genes in endothelial cells, the products of which promote the adhesion and extravasation of leukocytes across the endothelial barrier.

	MODULATION OF NF-B ACTIVATION IN LUNGS

TOP ABSTRACT INTRODUCTION MOLECULAR BIOLOGY OF NF-B BACTERIA-INDUCED NF-B... ROLE OF NF-B IN... MODULATION OF NF-B ACTIVATION... INTERACTIONS BETWEEN NF-B AND... SUMMARY REFERENCES

The regulation of inflammation by cytokines involves an intricate balance of pro- and anti-inflammatory mediators. The principal anti-inflammatory cytokines released in lung tissue include IL-1RA, IL-4, IL-6, IL-10, IL-11, IL-13, and TGF-. The soluble cytokine receptors TNF receptor p55, TNF receptor p75, and IL-1 receptor type 2; the membrane-bound IL-1 receptor type 2; and the IL-18 binding protein also have anti-inflammatory activities. However, only a few of these have been shown to involve in lung injury via interaction with NF-B.

Intrapulmonary deposition of IgG immune complexes in rats causes alveolar macrophage activation and results in neutrophil-dependent parenchymal cell injury as characterized by increased pulmonary vascular permeability and alveolar hemorrhage (61, 151). Studies (32, 126-128) have identified IL-6, IL-10, IL-1RA, and IL-13 as endogenous regulators in this model of inflammatory lung injury. Lentsch et al. (70) showed that exogenously administered IL-4, IL-10, or IL-13 greatly attenuated the lung injury induced by IgG immune complexes and demonstrated that both IL-10 and IL-13 inhibited nuclear localization of NF-B in alveolar macrophages and lung tissues in a manner associated with preserved expression of IB- protein. These findings suggest that IL-10 and IL-13 reduce lung inflammation by preventing degradation of IB-, thus inhibiting the activation of NF-B.

Schottelius et al. (119) showed that IL-10 functions to block NF-B activity at two levels, through 1) suppression of IKK activity and 2) inhibition of NF-B DNA binding activity. To address the mechanism of action of IL-10, Wang et al. (149) tested the effects of IL-10 on different transcription factors including NF-B, NF-IL-6, activator protein (AP)-1, AP-2, glucocorticoid receptor, cAMP response element binding protein (CREB), Oct-1, and Sp1. They found that IL-10 selectively prevented NF-B activation, with the effect occurring rapidly and in a dose-dependent manner. Moreover, the effect was correlated with the cytokine synthesis inhibitory activity of IL-10. IL-10 inhibited chemokine expression (MIP-1 and MIP-2) by inducing both mRNA destabilization and NF-B inhibition (130). However, IL-4, which also inhibited cytokine mRNA accumulation in monocytes, showed little inhibitory effect on LPS-induced NF-B activation (149). Unlike IL-10, IL-4 enhanced mRNA degradation and failed to suppress cytokine gene transcription. These data point to distinct roles for IL-10 and IL-4 in inhibiting cytokine production by different mechanisms.

TGF-, a pleiotropic cytokine/growth factor, is believed to play a critical role in the modulation of inflammatory events. DiChiara et al. (33) demonstrated that exogenous TGF-1 inhibited the expression of the proinflammatory adhesion molecule E-selectin in vascular endothelium exposed to inflammatory stimuli. This inhibitory effect occurred at the level of transcription of the E-selectin gene and was dependent on the action of Smad proteins, a class of intracellular signaling proteins mediating the cellular effects of TGF-1 (33). Furthermore, this work demonstrated that Smad-mediated effects in endothelial cells resulted from a competitive interaction between Smad proteins activated by TGF-1 and NF-B activated by inflammatory stimuli (such as cytokines or bacterial LPS). This interaction is mediated by the transcriptional coactivator CREB-binding protein (CBP) (33). Augmentation of the limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad-CBP interactions (via a dominant negative strategy) effectively antagonized the ability of TGF-1 to block proinflammatory E-selectin expression (33). These data demonstrate a potentially important interaction between TGF-1-regulated Smad proteins and NF-B that is regulated by inflammatory stimuli in endothelial cells.

	INTERACTIONS BETWEEN NF-B AND OTHER TRANSCRIPTION FACTORS

TOP ABSTRACT INTRODUCTION MOLECULAR BIOLOGY OF NF-B BACTERIA-INDUCED NF-B... ROLE OF NF-B IN... MODULATION OF NF-B ACTIVATION... INTERACTIONS BETWEEN NF-B AND... SUMMARY REFERENCES

NF-B can differentially regulate the expression of many genes involved in inflammatory processes with different functions, in different cell types, and at different times. This diversity arises from a delicately balanced network of protein-protein interactions. NF-B activity is determined not only through its regulated nuclear localization signal but also by the cellular context. NF-B interacts with a large number of heterologous transcription factors, and these interactions can select for specific NF-B subunits and thereby lead to greater transcriptional selectivity. Figure 3 is a hypothetical schema for possible mechanisms by which NF-B interacts with other transcription factors.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Hypothetical models for interaction between NF-B and another transcription factor (TF). A: NF-B binds to B site and regulates target gene transcription without interaction with the other TF. B: the other TF, except NF-B, binds to its specific binding sequence and initials gene transcription. C: NF-B and the other TF bind to their specific binding sites and synergistically result in enhanced gene transcription (+). D: the other TF can stimulate NF-B DNA binding and transactivation through B elements in the absence of other TF sites. E: interaction between NF-B and the other TF sometimes does not involve a precise promoter/enhancer organization or even require a B element. F: the other TF may negatively regulate NF-B transactivation ().

AP-1 is one such transcription factor involved in the control of many inflammatory mediators. AP-1 is composed of protooncogene products as heterodimers of c-Fos and c-Jun or as c-Jun-c-Jun homodimers. Activated AP-1 completely disrupts the structure of the nucleosome (99) and is considered to be the important first step in the chromatin remodeling process involved in the initial binding of transcriptional factors to a nucleosomal template. Interestingly, the interactions between NF-B and AP-1 sometimes do not involve a precise promoter/enhancer organization or even require a B element. Fos and Jun can stimulate Rel A DNA binding and transactivation through B elements in the absence of AP-1 sites, whereas Rel A can stimulate AP-1 DNA binding and activation through AP-1 sites in the absence of a B element (137). Armstead et al. (5) reported that tissue factor mRNA level and protein activity in plasma and lungs were markedly increased after trauma and the increase was AP-1 and NF-B activation dependent. However, other observations suggest that AP-1 and NF-B can function independently from each other. Shenker and Abraham (131) studied the transcriptional mechanisms involved in regulating pulmonary cytokine expression after hemorrhage and showed activation of NF-B and CREB but not of AP-1, CCAAT/enhancer binding protein- (C/EBP-), or Sp1 in lung mononuclear cells isolated from mice. Liu et al. (75) also observed that the expression of TNF- by NF-B was independent of c-Jun in primary human macrophages. A 120-bp TNF- promoter-reporter possessing binding sites for NF-B (B3), C/EBP-, and c-Jun was activated by cotransfection of plasmids expressing the wild-type version of each of these transcription factors. Dominant negative versions of NF-B p65 and c-Jun but not of C/EBP- suppressed LPS-induced TNF- secretion in primary human macrophages (75). NF-B p50/p65 heterodimers were bound to the B3 site and c-Jun was bound to the 103 AP-1 site of the TNF- promoter after LPS stimulation. By transient transfection, NF-B p65 and p50 were shown to synergistically activate the TNF- promoter. In contrast, such synergy was not observed between NF-B p65 or NF-B p50 and c-Jun or C/EBP- even in the presence of the coactivator p300 (75). This result suggests that adjacent B3 and AP-1 sites in the human TNF- promoter contribute independently to the LPS-induced activation response. More work is needed to determine how AP-1 interacts with other transcriptional factors such as NF-B and thereby controls the inflammatory responses resulting in acute lung injury.

The C/EBP family of transcription factors, together with AP-1 and activating transcription factor/CREB, belong to a class of DNA binding proteins termed bZIP proteins (146). These proteins are characterized by their leucine zipper structure and the adjacent DNA binding basic region, both located in the COOH-terminal portion of the proteins. Four members of the C/EBP family, C/EBP-, C/EBP-, C/EBP-, and C/EBP- (24), form homodimers and heterodimers with each other and bind with a similar affinity to various C/EBP binding sites (24, 66). Stein et al. (138) demonstrated a functional and physical association between NF-B and C/EBP. These associations are characterized by 1) inhibition of NF-B-induced expression of promoters containing NF-B binding sites by the expression of C/EBP family members and 2) positive synergistic action of NF-B family members with C/EBP family members on promoters containing C/EBP binding sites.

Evidence indicates that NF-B subunits act in concert with members of the C/EBP family of transcription factors in regulating gene expression mediated by the multimerized c-Fos serum response element (138). Several functional NF-B subunits (p50, p65, and c-Rel) interacted with three different isoforms of C/EBP (, , and ) (138), indicating an interaction between these families and not merely a limited interaction between individual members. In another study (136), it was shown that C/EBP repressed NF-B transactivation through B elements, whereas NF-B stimulated DNA binding and transactivation through C/EBP binding sites. The IL-8 promoter contains a region with binding sites for both NF-B and C/EBP in close proximity that is required for TNF-- and IL-1-mediated activation (136). NF-B and C/EBP cooperatively bind this element and thereby induce the expression of IL-8 (136). Thus C/EBP specifically modulates NF-B function and cooperatively stimulates the transcription of genes.

Binding elements for NF-B and CREB are present in the enhancer/promoter regions of the immunoregulatory cytokine genes IL-1 and TNF- and have important functions in modulating transcription of these genes (9, 125, 145). NF-B and CREB were activated in the lungs after endotoxemia or blood loss (16, 67, 131); however, precisely how these two transcription factors cooperate to control inflammatory gene transcription in acute lung injury remains unclear.

High-mobility group protein-1 (HMG-1) is a highly conserved protein with >95% amino acid identity between rodents and humans (80, 105). HMG-1 was initially identified as a nonhistone nuclear protein that binds to the narrow minor groove of the AT sequence-rich B form of DNA. HMG-1 has been implicated in the regulation of gene transcription and in stabilizing nucleosome formation (15, 52, 58, 80, 105). A study (148) showed that HMG-1 is a key late mediator of endotoxin lethality. HMG-1 given intratracheally produced acute inflammatory injury in the lungs, with neutrophil accumulation, development of lung edema, and increased pulmonary production of IL-1, TNF-, and MIP-2 (1). In endotoxin-induced acute lung inflammation, administration of anti-HMG-1 antibody either before or after endotoxin challenge decreased the migration of neutrophils into the lungs and prevented pulmonary edema. These protective effects of the anti-HMG-1 antibody were specific because pulmonary levels of IL-1, TNF-, or MIP-2 were not decreased after therapy with the anti-HMG-1 antibody (1), thus pointing to a role for HMG-1 as a distal mediator of acute inflammatory lung injury.

The experiments of Brickman et al. (22) revealed the interaction between HMG-1 protein and members of the NF-B family. They showed that the Drosophila protein DSP1, a HMG-1/2-like protein, binds DNA cooperatively with NF-B, the p50 subunit of NF-B, and the Rel domain of Dorsal (22). This cooperativity is also apparent with other DNA molecules bearing consensus Rel protein binding sites. Moreover, the cooperativity observed in these DNA binding assays is paralleled by interactions between protein pairs in the absence of DNA. It is apparent from the work thus far that further studies addressing the significance of interactions of HMG-1 with NF-B in the mechanism of acute lung injury will be important.

There is growing evidence that the regulation of gene expression is not mediated solely by the presence or absence of a particular set of transcription factors. The mechanisms regulating NF-B activation, NF-B by IB, and the interactions of NF-B with other transcription factors are complex. Additional studies that make functional sense of this complexity will not only enhance our understanding of the role played by transcription factors in mediating lung inflammation and acute lung injury but will also lead to novel therapies directed against lung inflammation.

	SUMMARY

TOP ABSTRACT INTRODUCTION MOLECULAR BIOLOGY OF NF-B BACTERIA-INDUCED NF-B... ROLE OF NF-B IN... MODULATION OF NF-B ACTIVATION... INTERACTIONS BETWEEN NF-B AND... SUMMARY REFERENCES

Transcriptional activities in which the NF-B family members play a central position are key to understanding the pathobiology of acute lung injury. Therefore, modification of transcription becomes a logical therapeutic target for acute lung injury. Activation of NF-B and other transcription factors represents a complex network of finely balanced protein-protein interactions. The balance of proinflammatory to anti-inflammatory cytokines is regulated by a highly intricate network of transcription factors. Under pathological conditions, anti-inflammatory mediators provide insufficient control over proinflammatory activities. Despite the complexities inherent in the human immune response, therapeutic intervention with specific inhibitors of transcription signaling may have significant benefits in lung injury. Future studies in this area are likely to lead to novel therapies for the wide range of NF-B-mediated inflammatory diseases including acute lung injury.