key: cord-0005726-5xw6ceoq authors: Christman, J. W.; Lancaster, L. H.; Blackwell, T. S. title: Nuclear factor k B: a pivotal role in the systemic inflammatory response syndrome and new target for therapy date: 1998 journal: Intensive Care Med DOI: 10.1007/s001340050735 sha: 2a1151548cb16fb9bf2c0571f1e3c89f75bc8b3e doc_id: 5726 cord_uid: 5xw6ceoq nan Pathogenic stimuli that result in the systemic inflammatory response syndrome (SIRS) cause a broad range of host responses that include the production of protein and lipid mediators, expression of cell surface receptors and adhesion molecules, induction of enzymes and production of acute phase proteins, as well as activation of inflammatory cells. SIRS, which is referred to as sepsis when the inflammatory response is induced by infection, can lead to dire consequences, including multiple organ dysfunction syndrome (MODS), the acute respiratory distress syndrome (ARDS) and death. ARDS is a prototypic acute inflammatory lung disease and MODS associated with SIRS is characterized by diffuse organ damage with prominent neutrophilic inflammation. The paradox of SIRS is that host immune responses are critical for defense against infection, but excessive or dysregulated inflammation seems to result in neutrophil-mediated tissue injury and organ dysfunc-tion. Understanding the molecular events that regulate neutrophilic inflammation could facilitate specific intervention to prevent host tissue injury in SIRS without substantially compromising the host defense. In SIRS, neutrophilic inflammation appears to be the result of the local production of cytokines, chemokines, endothelial-leukocyte adhesion molecules and enzymes, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), whose production is regulated by the ubiquitous transcription factor complex NF-kB (Table 1) . NF-kB is a DNA binding protein necessary for directing high level transcription of many proinflammatory genes in tissue culture; however, the extent to which NF-kB controls specific biological processes in vivo is still being investigated. In this review, we discuss briefly the relevant molecular biology of NF-kB, including the process of activation of NF-kB and the role of NF-kB in the production of cytokines and other pro-inflammatory molecules. Within this context, we summarize information about NF-kB in human disease and focus on clinically relevant treatment approaches that could attenuate activation of NF-kB and Table 1 Human pro-inflammatory molecules that are regulated by NF-kB Colony stimulating factors G-CSF GM-CSF Interleukins IL-1b IL-2 IL-6 IL-12 diminish the expression of pro-inflammatory genes, potentially limiting tissue injury and inflammation and improving the outcome of SIRS. The Rel/NF-kB family of transcription factors represents a distinct paradigm of nuclear transactivating factors whose activity is inducible via control of its nuclear localization. These enhancer binding proteins are sequestered in the cytoplasm by inhibitory molecules, termed IkBs. IkB is destroyed upon cell stimulation, and the kB factors enter the nucleus and associate with their cognate DNA binding sites, initiating gene transcription. The Rel/NF-kB family of transcriptional transactivators is a ubiquitous multiprotein complex specialized for rapid response of the cell to a wide variety of both normal and pathogenic agents [1] including those stimuli that are involved in the pathogenesis of SIRS, MODS, and ARDS (Table 2) . NF-kB acts as a transcription factor by binding to a decameric DNA sequence motif found in the promoters and introns of several genes, including those of the immunoglobulin k light chain, the human immunodeficiency virus long terminal repeats, numerous cytokines, chemokines, adhesion molecules, enzymes and growth factor receptors. The molecular and biochemical nature of the Rel/ NF-kB complex has been well reviewed [1±4] and will only be discussed in this review in a condensed form. Authentic NF-kB is a heterodimer consisting of a 50 kD polypeptide (nfkB1 = p50) [5] and a 65 kD polypeptide (RelA = p65) [6]. Other Rel family proteins include c-Rel and p52 (nfkB2) which, along with p50 and RelA, can form various combinations of homodimers and heterodimers that regulate specific target gene transcription. The carboxy terminal domains of RelA(p65) and c-Rel contain strong transcriptional transactivation regions [7] . By contrast, p50 homodimers bind to DNA but are thought to block transcription since it lacks a transactivation domain [8] . IkB was first described as a cytoplasmic protein which inhibits the DNA binding activity of the heterodimeric NF-kB complex [9,10]. In the cytosol, IkB proteins form complexes with heterodimeric NF-kB and can be inactivated following stimulation of cells with a wide variety of distinct agents including lipopolysaccharide (LPS), TNFa, IL-1b, phorbol esthers, growth factors, viral proteins and ultraviolet light. Some of these stimuli, endotoxemia, cytokinemia, ischemia/reperfusion, hyperoxia and mechanical stress, are probably relevant to the pathophysiology of SIRS, MODS and ARDS. Like their binding partners (Rel/NF-kB proteins), the IkBs are encoded by a small multigene family [1, 4] , which includes IkB-a (MAD3, pp40, RL-IF1, ECI), IkB-b, IkB-g and BCL-3. Recently, the signalling pathways from specific receptors to activation of NF-kB have been worked out for both TNF-a and IL-1b [11±14]. TNF-a activates NF-kB primarily through binding to the type 1 TNF receptor (TNFR1), which then induces a signal transduction cascade through several intermediate signalling proteins, resulting in activation of NF-kB-inducing kinase (NIK), a member of the mitogen-activating protein (MAP) kinase kinase kinase (MAP3 K) family [11] . When TNF-a binds to the TNFR1, there is an association between the cytoplasmic domain of TNFR1 and the TNFR1-associated death domain protein (TRADD), the receptor-interacting protein (RIP), and the TNF receptor-associated factor-2 (TRAF-2) that forms an active signalling complex that interacts with NIK. Activation of NIK results in phosphorylation of IkB kinases (IKK), which binds to and phosphorylates IkB-a at serines 32 and 36 [11, 12] . Phosphorylated IkBa is targeted for destruction by the ubiquitinization/proteasome (26 S) degradation pathway, allowing the translocation of NF-kB to the nucleus [1, 15, 16] . IL-1b binding to its receptor also results in NIK and IKK activation, followed by IkB-a degradation [17±20]. IL-1b binds to the type 1 IL-1 receptor (IL-1R1) and the IL-1 receptor accessory protein (IL-1RAcP) facilitates an interaction between IL1 receptor-associated kinase (IRAK) and TNF receptor-associated factor-6 (TRAF-6) that results in activation of NIK, IKK and NF-kB. In combination, these data seems to indicate that NIK is a common mediator in the NF-kB signalling cascade that results from receptor mediated TNF and IL-1 stimulation. Although it is likely that activation by endotoxin (LPS) follows a similar scheme, the specific link to NIK has not yet been established. A simplified version of the TNFa and IL-1b pathways is illustrated in Fig. 1 . Substantial in vitro data suggest that activation of NF-kB is a critical proximal step in the inflammatory response. In addition, several studies have demonstrated a link between in vivo NF-kB activation with cytokine production and the generation of inflammation in animal models of inflammatory diseases [21±27]. Investigations regarding the role of NF-kB in humans have only recently been undertaken. In the peripheral blood monocytes of patients with sepsis, NF-kB activation predicts mortality [28] . Specifically, all the patients in this study who died from sepsis had increased NF-kB activation (greater than twice baseline) in the first 6 days, whereas all the patients who survived had NF-kB activation that remained less than twice baseline at each time point during the 14-day study period. Also, NF-kB is activated in the alveolar macrophages from patients with ARDS to a significantly higher degree than in alveolar macrophages from critically ill patients with other diseases [29] . This finding correlates with previous reports that IL-8 and TNFa are increased in lung lavage in patients with ARDS [30±31]. Patients with other inflammatory diseases, including rheumatoid arthritis and Crohn's disease have been shown to have increased NF-kB activation. In the synovium of patients with rheumatoid arthritis, NF-kB activation is increased compared to patients with osteoarthritis [32]. In patients with Crohn's disease, NF-kB activation in bowel sections is increased compared with controls [33]. In addition, NF-kB has been implicated in the pathogenesis of several human malignancies [34] and in Alzheimer's disease [35] . Experimental evidence linking NF-kB activation in specific cells or tissues with other inflammatory diseases in humans is lacking but should be forthcoming. If NF-kB activation proves to be an important determinant of systemic inflammation, interventions designed to limit NF-kB activation could be beneficial in SIRS and other inflammatory diseases. Currently, no clinical studies have used inhibition of NF-kB activation as a goal of therapy; however, several currently available therapeutic approaches could be used to target NF-kB. Although blocking NF-kB activation is likely to inhibit neutrophilic inflammation and diminish organ injury secondary to exuberant cytokine production, it is uncertain how this treatment approach would affect host defense functions. In future, it may be important to design clinical trials in SIRS and ARDS with the goal of blocking NF-kB activation, either in tissue samples or in bronchoalveolar lavage or white blood cells. If NF-kB activation can be effectively inhibited, the results of NF-kB inhibition on clinical outcome can be assessed. One strategy for blocking NF-kB activation in SIRS is to prevent the interaction of TNFa, IL-1b or endotoxin with responding cells. The proximal cytokines, as well as endotoxin, are fully capable of producing a sepsis-like syndrome when injected into animals or man and are known to stimulate NF-kB activation in a variety of cell types in vitro. Agents that block TNFa, IL-1 and endotoxin, such as anti-TNF antibodies, soluble TNF receptors, IL-1 receptor antagonist, anti-endotoxin antibodies and soluble CD14, could limit NF-kB activation and prevent inflammation and organ dysfunction in SIRS. Although these agents have been shown to be ef-1133 Fig. 1 TNF-a binds to the type 1 TNF receptor (TNFR1), which results in an association with TNFR1-associated death domain protein (TRADD), the receptor-interacting protein (RIP), and the TNF receptor-associated factor-2 (TRAF-2). These cytoplasmic proteins form an active signalling complex that interacts with NF-kB-inducing kinase (NIK). Activation of NIK results in phosphorylation of IkB kinases (IKK), which cause phosphorylation IkB. Phosphorylated IkB is targeted for destruction by the ubiquitinization/proteasome degradation pathway, allowing the translocation of NF-kB to the nucleus. IL-1b binds to the type 1 IL-1 receptor (IL-1R1) and the IL-1 receptor accessory protein (IL-1RAcP) which facilitates an interaction between IL1 receptor-associated kinase (IRAK) and TNF receptor-associated factor-6 (TRAF-6). These proteins form an active signalling complex that also results in activation of NIK and IKK leading to the sequence of events that results in activation of NF-kB. Activation of NF-kB results in expression of mRNA of a variety of pro-inflammatory mediators (see Table 1 ) which are involved in the pathogenesis of SIRS, MODS and ARDS. IkB is also induced by NF-kB activation and contributes to the down-regulation of this intracellular signalling cascade fective in limiting mortality and improving the pathophysiology of experimental SIRS, none has been shown to be efficacious in human studies. Blocking a single mediator, especially after the initiating insult, might be insufficient to inhibit NF-kB significantly because of the redundancy of proximal mediators with the potential to activate NF-kB. In vitro studies have shown that blocking the individual cytoplasmic components of the TNFa and IL-1b activation pathways, including TRADD, RIP, TRAF-2 and TRAF-6, is an effective way to block activation of NF-kB. Strategies that manipulate these intracellular signalling molecules have not been tried in animal models and are far from clinical trials in human diseases. Furthermore, none of these molecules appears to be both a critical and essential pathway for activation of NF-kB. Several recent reviews, including our own, have addressed many of these anti-cytokine strategies and their limitations [36±37]. Here, we discuss several potential treatment strategies that suppress NF-kB activation by a variety of stimuli and have the potential globally to down-regulate acute inflammation in SIRS (Table 3) . While some of these approaches are already feasible (antioxidants, corticosteroids and induction of endotoxin tolerance), more specific treatments (inhibition of NIK and IKK, and the proteosome inhibitors) will be dependent on advances in technology and close cooperation between pharmaceutical companies and clinical scientists. In terms of NF-kB inhibition, antioxidants are the best studied class of agents. Antioxidants have been investigated as inhibitors of NF-kB activation because the generation of reactive oxygen species (ROS) is postulated to be a vital link in mediating NF-kB activation by a variety of stimuli. Four lines of evidence support the role of ROS in NF-kB activation. [46] and S-allyl cysteine (SAC) [47] . Since ROS appear to be important intermediates in NF-kB activation, inhibiting this step might be beneficial in limiting inflammation in certain clinical settings. Several recent early clinical trials suggest that NAC and related compounds may be an effective treatment for ARDS, and it is possible that this benefit has been derived through blocking the activation of NF-kB [48±50]. Studies in animal models of ARDS have indicated that effects of NAC have a complex dose-response relationship where low doses improve, but high doses worsen, parameters of lung injury [51] . Enormous efforts have been made to characterize the intracellular signalling process that leads to the activation of NF-kB [52±60]. The critical common pathway that leads to activation of NF-kB, activation of IKK by NIK, is rapidly being reported in the literature at an unprecedented pace. Two specific IkB kinases (IKKa and IKKb) have been identified [11±13], which phosphorylate IkBa on serine 32 and 36 that is required for processing via the ubiquitin-proteasome pathway. Overexpression of either an inactive IKKa or IKKb mutant associates with both IkBa and NIK and blocks activation of NF-kB by TNFa and IL-1b [11, 13] . These data seem to indicate that the IkB kinase complex, termed the IkB kinase (IKK) signalsome, involves aggregation of two distinct kinases, IKKa and IKKb, that are activated by an interaction with NIK [61±62]. An inactive NIK mutant has been shown to block activation of NF-kB TNFa and IL-1b, which suggests NIK is a critical step that leads to activation of the IKK signalsome [63] . Recently, it has been shown that NIK activates the IKK signalsome by phosphorylating IKKa, but not IKKb, at serine 176 [14] . Currently there is a frenzied search for compounds that specifically block or antagonize NIK and/or the IKK signalsome since these agents could have broad clinical applications for the treatment of a wide variety of inflammatory diseases that include SIRS, MODS and ARDS. It is likely that a highly specific blockade of NIK and the IKK signalsome will be required because the use of less specific kinase blockers has yielded conflicting results [52±60]. Table 3 Agents which block activation of NF-kB and might be effective treatments for SIRS, MODS, and ARDS · Antioxidants · Inhibition of NF-k B inducing kinase (NIK) · Inhibition of the I-k B kinase (IKK) signalsome · Proteasome inhibitors · Corticosteroids · Induction of endotoxin tolerance The 26 S proteasome degrades IkB-a after it is targeted by phosphorylation and ubiquitinization. Several studies have shown that inhibitors of the proteasome complex block NF-kB activation and nuclear translocation in cell culture [64±68] . Recently, certain proteasome inhibitors have been given to rodents without significant toxicity. Administration of the proteasome inhibitor calpain inhibitor 1 by intraperitoneal injection in rats suppressed expression of two NF-kB-dependent genes, iNOS and COX-2 [69] . Chemical proteasome inhibition will probably affect other cellular processes in addition to NF-kB activation, such as antigen processing, cell cycle regulation and the processing of other transcription factors [70] . However, based on available in vitro data, proteasome inhibitors appear to be acceptable agents to suppress NF-kB activation and could be beneficial for short-term treatment to limit inflammation if toxicity could be overcome. Corticosteroids are a group of compounds with a broad range of effects on the immune system, and have long been known to be potent anti-inflammatory agents and potent in vitro suppressors of cytokine production [71] . Some of the anti-inflammatory effects of corticosteroids are mediated through inhibition of NF-kB activation. Corticosteroids have been shown to block NF-kB activation in vitro in two ways. First, glucocorticoid receptors can interact directly with RelA, the transactivating component of NF-kB, to inhibit DNA binding [72] . More recently, two separate groups of investigators have shown that dexamethasone, a potent corticosteroid, increases the expression of IkB-a mRNA and synthesis of functional IkB-a protein [73, 74] . Although further research is necessary, inhibition of DNA binding of RelA and stabilization of cytoplasmic NF-kB by a relative excess of newly synthesized IkB may be an effective dual mechanism by which steroids block the synthesis of cytokines. Although corticosteroids have not proven to be beneficial in post-treatment studies of patients with SIRS and ARDS, sufficient inhibition of NF-kB may not have been achieved, and glucocorticoids may have other effects that are detrimental in this condition. Endotoxin tolerance refers to a phenomenon where by an endotoxin-triggered response is at least partially abrogated by prior exposure to endotoxin. Many investigators have reported that tolerance can be induced in both animal models and human volunteers by treatment with repeated small doses of endotoxin. A consistent observation is that endotoxin-tolerant macrophages have blunted gene expression of cytokines, including TNFa, IL-1b, IL-6 and IL-8, in response to treatment with endotoxin. Endotoxin-tolerant cells are cross tolerant to both TNFa and IL-1b, suggesting that endotoxin tolerance is a fundamental process that occurs at the level of the cell. We have recently investigated the effect of induction of endotoxin tolerance on NF-kB-dependent responses by utilizing a rat model of neutrophilic lung inflammation. Rats were rendered endotoxin-tolerant by four daily injections of low dose endotoxin. When endotoxin-tolerant rats were treated with high dose intraperitoneal endotoxin injection, they had decreased NF-kB activation and chemokine gene expression in lung tissue, as well as attenuated neutrophilic lung inflammation compared to endotoxin-sensitive rats [75±76] . Studies in a rat alveolar macrophage cell line indicate that the mechanism of the tolerance effect is related to depletion of RelA [77] . Other investigators have shown a second mechanism of in vitro endotoxin tolerance: increased gene expression of p105 that results in excess production and homodimerization of p50 [78] . This has the overall effect of preventing gene activation, since p50 homodimers do not bind to IkB and can bind to the decameric NF-kB DNA sequence, but lack a transactivating domain. Exploiting the natural phenomenon of endotoxin tolerance may be an effective way to suppress NF-kB-dependent inflammation. Several studies in animal models and clinical trials have suggested that the induction of tolerance might be effective in selected patients with SIRS [79±81]. These data lead us to believe that induction of endotoxin could be used to prevent or relieve at least some sequelae of gram-negative sepsis through inhibition of NF-kB-dependent inflammatory gene expression. NF-kB is an important transcription factor complex that appears to play a fundamental role in regulating acute inflammation through activation of the cytokine cascade and production of other pro-inflammatory mediators. There is increasing evidence that NF-kB is important in the pathobiology of disease states such as SIRS, MODS and ARDS; therefore, therapeutic interventions aimed at limiting NF-kB activation and down-regulating production of inflammatory mediators could prove to be beneficial in decreasing host-derived tissue injury and organ dysfunction. Specific interventions that hold promise for suppressing NF-kB activation include the use of antioxidants, inhibition of NIK and the IKK signalsome, treatment with proteasome inhibitors, induction of endotoxin tolerance and, possibly the use of corticosteroids in selected patients. 4. Nolan GP, Baltimore D (1992) The inhibitory ankyrin and activator rel proteins. Curr. Opin. Genet.Dev. 2: 211±220 5. Ghosh S, Gifford AM, Riviere LR, Tempst P, Nolan GP, Baltimore D Rel/NF-kB/IkB family: intimate tales of association and dissociation Malignant transformation by mutant Rel proteins Signal transduction: the nuclear target Inhibition of NF-kappa B activation by dimethyl sulfoxide correlates with suppression of TNF-alpha formation, reduced ICAM-gene transcription, and protection against endotoxininduced liver injury S-allyl cysteine inhibits activation of nuclear factor kappa B in human T cells N-acetylcysteine enhances recovery from acute lung injury in man. A randomized, double-blind, placebo-controlled clinical study Antioxidant treatment with N-acetylcysteine during adult respiratory distress syndrome: a prospective, randomized, placebo-controlled study A trial of antioxidants N-acetylcysteine and procysteine in ARDS. The Antioxidant in ARDS Study Group Low-dose N-acetylcysteine protects rats against endotoxin-mediated oxidative stress, but high-dose increases mortality Redox regulation of NF-kappa B activation Signalinduced degradation of I kappa B alpha requires site-specific ubiquitination N-and C-terminal sequences control degradation of MAD3/I kappa B alpha in response to inducers of NFkappa B activity Activation of NF-kappa B in vivo is regulated by multiple phosphorylations Activation of NF-kappa B by phosphatase inhibitors involves the phosphorylation of I kappa B alpha at phosphatase 2A-sensitive sites Studies into the effect of the tyrosine kinase inhibitor herbimycin A on NF-kappa B activation in T-lymphocytes. Evidence for covalent modification of the p50 subunit Tyrosine phosphorylation of I kappa Balpha activates NF-kappa B without proteolytic degradation of I kappa B-alpha Protein tyrosine phosphatase inhibitors block tumor necrosis factor-dependent activation of nuclear transcription factor NFkappa B Activation of the IkBa kinase complex by MEKK1, a kinase of the JNK pathway IKK and IKK-: cytokineactivated IkappaB kinases essential for NF-kappaB activation The I-kappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation Tumor necrosis factor (TNF) receptor 1 signalling downstream of TNF receptor-associated factor 2. Nuclear factor kappaB (NFkappaB)-inducing kinase requirement for activation of activating protein 1 and NFkappaB but not of c-Jun N-terminal kinase/stress-activated protein kinase Activation of NF-kB requires proteolysis of the inhibitor IkB-a: signal-induced phosphorylation of IkB-a alone does not release active NF-kB Signal-induced site-specific phosphorylation targets IkBa to the ubiquitin-proteasome pathway Inhibition of IkB-alpha and IkB-beta proteolysis by calpain inhibitor I blocks nitric oxide synthesis Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kB N-acetyl-leucinyl-leucinyl-norleucinal inhibits lipopolysaccharide-induced NF-kB activation and prevents TNF and IL-6 synthesis in vivo Effect of calpain inhibitor I, an inhibitor of the proteolysis of IkB, on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat The ubiquitinproteasome pathway Ozone induction of cytokine-induced neutrophil chemoattractant (CINC) and nuclear factor-kappa b in rat lung: inhibition by corticosteroids Physical association and functional antagonism between the p65 subunit of transcription factor NF-kB and the glucocorticoid receptor Role of transcriptional activation of IkBa in mediation of immunosuppression by glucocorticoids Immunosuppression by glucocorticoids: inhibition of NF-kB activity through induction of IkB synthesis Endotoxin tolerance: in vivo regulation of tumor necrosis factor and interleukin-1 synthesis is at the transcriptional level Impaired activation of NF-kB in endotoxin tolerant rats is associated with down-regulation of chemokine gene expression and inhibition of neutrophilic lung inflammation Endotoxin tolerance is associated with depletion of RelA and increased formation of p50 homodimers in rat alveolar macrophages Tolerance to lipopolysaccharide involves mobilization of nuclear factor kappa B with predominance of p50 homodimers Monophophoryl Lipid A attenuates the effect of endotoxin shock in pigs Comparison of the induction of endotoxin tolerance in endotoxemia and peritonitis by monophosphoryl Lipid A and lipopolysaccharide Pretreatment of normal humans with monophosphoryl lipid A induces tolerance to endotoxin: a prospective, double-blind, randomized, controlled trial