NF-κB and the innate immune response

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In the innate immune reaction, microbial pathogens activatephylogenetically conserved cellular signal transductionpathways that regulate the ubiquitous nuclear factor-κB(NFκB). NF-κB has pleiotropic functions in immunity; however,it is also critical for development and cellular survival. Manyaspects of how the different pathways utilize a common kinasecomplex that ultimately activates NF-κB have been clarified bygene inactivation and biochemical analysis.

AddressesMax-Delbrück-Center for Molecular Medicine, MDC Robert-Rössle-Strasse 10, 13122 Berlin, GermanyCorrespondence: Claus Scheidereit;e-mail: [email protected]

Current Opinion in Immunology 2000, 12:52–58

0952-7915/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved.

Abbreviationsββ-TrCP β-transducin-repeat-containing proteinECSIT evolutionarily conserved signaling intermediate in Toll

pathwaysERK extracellular-signal-regulated kinaseHOS homologous to SlimbIKAP IKK-associated proteinIκκB inhibitor of NFκBIKK IκB kinaseIL-1R IL-1 receptorIRAK IL-1R-associated kinaseJNK c-Jun amino-terminal kinaseLPS lipopolysaccharideMAP3K mitogen-activated protein kinase kinase kinaseMEKK1 MAPK/ERK kinase kinase 1MyD88 myeloid differentiation factor 88NEMO NF-κB essential modulatorNF-κκB nuclear factor κBNIK NF-κB-inducing kinaseTAB1 TAK1-binding protein 1TAK1 transforming growth factor β activated kinase 1TLR Toll-like receptorTNFαα tumor necrosis factor αTPL-2 tumor progression locus 2TRAF-6 TNF receptor associated factor 6

IntroductionAs an immediate host defense reaction, the innate immuneresponse involves secretion of cytokines and other mediatorsleading to an inflammatory process that has antimicrobialeffects. It is well documented that phylogenetically con-served signaling mechanisms in innate immunity providingan immediate cellular reaction utilize the nuclear factor κB(NF-κB) system at the heart of this first-line defense. NF-κBfamily members contain a conserved DNA binding anddimerization domain called the Rel homology domain.Mammalian cells contain five NF-κB subunits — RelA(p65), c-Rel, RelB, p50 and p52 — which form various het-ero- and homo-dimers. The p50 and p52 subunits, whichlack transactivation domains, are produced by processing ofprecursor molecules of 105 kDa and 100 kDa, respectively.

NF-κB is rapidly activated by a large spectrum of chemical-ly diverse agents and cellular stress conditions includingbacterial lipopolysaccharides (LPSs), microbial and viralpathogens, cytokines and growth factors [1]. Activationinvolves liberation of NF-κB dimers from cytoplasmic com-plexes with ankyrin-repeats containing IκB (inhibitor ofNF-κB) proteins, following phosphorylation of IκBs by thecytokine-responsive IκB kinases IKKα and β, ubiquitinationof phosphorylated IκBs by a ubiquitin ligase complex anddegradation by the 26S proteasome [2]. NF-κB is itself a crit-ical transcriptional activator of cytokines involved in theinnate immune response, including TNFα and IL-1. Inaddition to its prominent role in innate immunity, NF-κBexerts important functions in the adaptive immune system.Gene-inactivation studies of single NF-κB members haverevealed that Rel proteins are required for lymphocyte acti-vation by controlling proliferation, immunoglobulin isotypeswitching and expression of cytokines and their receptors[3,4]. Furthermore, other crucial cellular functions of NF-κBare not confined to the immune system. NF-κB serves toprotect against apoptosis and supports cell cycle progression[5]. As all these different functions are exerted by NF-κBcomplexes, a current challenge is to determine how pathwayspecificity is reached. Within the past year further insight hasbeen gained by gene ablation experiments in mice and bythe use of transfected signaling molecules. These experi-ments brought on a better characterization of thecomposition and function of the IKK complex, as well as themolecules involved in its activation cascade, which form thescope of this review.

The IκκB kinases IKKαα and IKKββ have verydistinct functionsTwo years ago, the kinases that phosphorylate IκBs at twoamino-terminal serine residues (Ser32 and Ser36 for IκBα,Ser19 and Ser23 for IκBβ) in response to LPS, TNFα, IL-1,PMA (phorbol 12-myristate 13-acetate) or HTLV-1 Tax wereidentified as IKKα and IKKβ (for a review, see [6]). IKKαand IKKβ contain closely related kinase domains, leucinezippers and helix-loop-helix regions, and form heterodimers,which are part of a large ~800 kDa complex. Biochemical andtransfection experiments suggested non-equivalent func-tions of IKKα and β. IKKβ is a more potent NF-κB activatorand has a higher kinase activity towards IκBα. More dramat-ic differences became obvious by the generation of IKKα-and IKKβ-deficient mice (Figure 1). IKKα–/– mice present-ed an unexpected phenotype including shorter limbs andskull, and a fused tail, all enveloped in a shiny and sticky skin[7••–9••]. These mice die perinatally and have hyperprolifer-ative epidermal cells that do not differentiate; however, IL-1-and TNFα-induced NF-κB activation is normal, and so isphosphorylation and degradation of IκBα and IκBβ. Thus,IKKα is involved in dermal and skeletal development andcannot be compensated for by IKKβ.

NF-κκB and the innate immune responseEunice N Hatada, Daniel Krappmann and Claus Scheidereit*

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NF-κκB and the innate immune response Hatada, Krappmann and Scheidereit 53

The skin defect in IKKα–/– mice resembles that seen intransgenic keratinocytes expressing a dominant-negativeIκBα mutant devoid of IKK phosphorylation sites [10].Given that the opposite effect — a markedly thin epider-mis — was seen in transgenic mice overexpressing p50, thenon-transactivating NF-κB subunit [10], it is likely thatNF-κB family members are involved in growth control instratified epithelium. However, the present data do notreveal which NF-κB and IκB species, and perhaps otherIKK substrates, are involved. The next challenge will be toidentify the developmental signals that activate IKKα.

IKKβ-defective mice die as embryos and show massiveliver degeneration due to hepatocyte apoptosis, a pheno-type similar to that of p65–/– mice [11••–13••]. NF-κBactivation by IL-1 or TNFα is strongly impaired but notcompletely abolished. This demonstrates that IKKβ is cru-cial for NF-κB activation but also suggests that IKKα orother presently unknown kinases weakly contribute thisaction. In the IKKα–/– or IKKβ–/– mutant mice each of theIKKs forms homodimers and can interact with IKKγ/ΝΕΜΟ(NF-κB essential modulator; see below) in the absence ofthe other [9••,11••]; however, it is not known if homodimersplay a normal physiological role. As the distinct phenotypesobserved in IKKα- and IKKβ-deficient mice suggest thateach kinase responds to different stimuli, specific adapterproteins or upstream kinases should exist allowing each ofthe IKKs to perform different tasks.

Composition of the IKK complexThe IKKs are part of a high molecular weight complex,which might contain regulatory subunits. One such com-ponent, NEMO/IKKγ, was cloned by geneticcomplementation of an HTLV-1-Tax-transformed ratfibroblast cell line [14••] and by biochemical purification[15,16]. IKKγ is essential for NF-κB activation by LPS,TNFα, IL-1, Tax, or phorbol 12-myristate 13-acetate.Deletions of either the amino terminus or carboxyl termi-nus of IKKγ block NF-κB activation [15,16]. IKKγ iscomposed of coiled-coil motifs — including a leucine zip-per — through which it presumably recruits upstreamactivators to the IKK complex. Interestingly, in IKKγ-deficient cells the apparent molecular weight of the IKKcomplex is 300–450 kDa instead of ~800 kDa [14••].Although determination of molecular weights by sizeexclusion chromatography must be interpreted with cau-tion, this might indicate the presence of further unknowncomponents.

Another molecule, IKAP (IKK-associated protein), wassuggested to be a scaffold protein able to bind to NF-κB-inducing kinase (NIK) and IKKs and to assemble theminto an active kinase complex in transfection assays [17].Overexpression of IKAP inhibits TNFα- and IL-1-induced NF-κB reporter activity, supposedly by titratingout components of the IKK complex; however, it remainsto be established whether IKAP is part of the same~800 kDa IKK complex containing IKKγ.

Recently, a new serine/threonine kinase IKK-i [18], whichshares 30% overall identity with IKKα or IKKβ, was shownto phosphorylate IκBα preferentially at Ser36 and to stimu-late an NF-κB reporter gene. But the IKK-i kinase activitywas not regulated by any known NF-κB stimuli. AlthoughIKK-i mRNA is strongly induced by LPS and inflammatorycytokines, it remains to be seen if this kinase is a physiolog-ical activator of NF-κB. Nevertheless, the discovery ofIKK-i does raise the question whether other IKK-relatedkinases exist that take part in NF-κB activation.

Activation of the IKK complex: formation ofreceptor complexesActivation of the IKK complex is required for NF-κB sig-naling in response to all inducers tested [2] with theexception of UV light, which can utilize a different path-way [19,20]. The most intensively studied IKK-activatingpathways include those originating from TNFα and IL-1receptors (Figure 2). The IL-1 receptor (IL-1R) is evolu-tionarily conserved with the Toll-like receptor (TLR)family, which mediates the innate immune response invertebrates and insects. Signaling via TLRs promotesdefense against microbial infection through activation ofNF-κB. Recent genetic and biochemical experiments havehighlighted the critical role of several signal mediators.

Figure 1

Defects in mice with homozyous inactivation of IKKα or IKKβ.Resulting IKK complexes in IKKα or β knockouts are depicted incomparison to wild-type complexes. Morphogenic signals need thepresence of the IKKα subunit. The nature of the upstream signal forIKKα to regulate keratinocyte differentiation and skeletonmorphogenesis remains to be established. Inflammatory cytokines (IL-1and TNFα) and bacterial LPS signal through IKKβ to activate NF-κBand absence of IKKβ results in embryonal death.

α βα βα β

IKKβ–/– mice IKKα–/– miceWild-typemice

LPS IL-1 TNFαDevelopmentalsignals

Defective keratinocyteproliferation anddifferentiationLimb and skeletonabnormalities

Defective responseto inflammatorycytokinesLiver cell apoptosis

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Upon binding of IL-1 to the IL-1R or LPS to TLRs viaCD14, a number of molecules are recruited to the receptorto mediate NF-κB activation. First, the adaptor moleculeMyD88 (myeloid differentiation factor 88) binds to thereceptor and interacts through its death domain with theprotein kinase IRAK (IL-1R-associated kinase), which inturn recruits the adaptor TRAF-6 (TNF-receptor-associat-ed factor 6) to the receptor complex. The importance ofMyD88 for IL-1 signaling was confirmed by analysis ofMyD88-deficient mice, which display defects in NF-κBactivation in response to the structurally similar cytokinesIL-1 or IL-18 [21]; moreover, MyD88–/– mice are highlyresistant to LPS-induced shock [22]. In contrast to IL-1 andIL-18 induction, however, c-Jun amino-terminal kinase(JNK) and NF-κB were activated in response to LPS,although with delayed kinetics. Thus, MyD88 is essentialfor the IL-1 and IL-18 pathways but LPS must act in addi-tion through a different mediator to activate NF-κB.

The serine/threonine kinase IRAK, upon recruitment tothe IL-1R complex, becomes phosphorylated and subse-quently interacts with TRAF-6. In IRAK-deficientembryonic fibroblasts, a significant reduction of theNF-κB response was observed upon IL-1 stimulation[23]. It was suggested that other IRAK homologs couldcompensate for the deficiency and lead to the partial NF-κB activation in these mice.

TRAF-6 has been implicated in NF-κB activation by IL-1and CD40 but not by TNFα. Now, Lomaga et al. [24] haveshown that deletion of the TRAF-6 gene in mice results inloss of NF-κB activation not only by IL-1 and CD40 butalso by LPS stimulation. TRAF6–/– mice are osteopetrotic,with defects in bone remodeling and tooth eruption as aresult of deficient osteoclast function. This raises the ques-tion of how bone differentiation signals are initiated byIL-1 or CD40, and what the role of NF-κB is in the process.

54 Innate immunity

Figure 2

Simplified, hypothetical overview of signalnetworks emanating from different receptorsand directed towards NF-κB. Ligand bindinginduces recruitment of TRADD (TNF-receptor-associated death domain), TRAF-2and RIP (receptor-interacting protein) toTNF-receptor 1 (TNF-R1) whereas IL-1R,IL-18 and LPS receptors (TLR and CD14)each attract MyD88, IRAK, TRAF-6 andECSIT. Further downstream are MAP3K-related kinases which are thought to linkreceptor-complexes and stimulate an IκBkinase (IKK) complex. TAK1 kinase and itsactivator TAB1 bind to TRAF-6 after IL-1stimulation and both interact with NIK. Cot(also known as TPL-2) is activated byTCR–CD3 and CD28 co-stimulation. Cotinteracts with both NIK and IKKα andactivates NIK. NIK can be directlyphosphorylated by TAK1 and Cot. TheMAP3Ks MEKK1, 2 and 3 all activate IKKswhen overexpressed. NIK can bind physicallyto TRAFs and to IKKα and is the only one ofthe MAP3K-related kinases shown whichdoes not activate JNK. NIK and MEKK1phosphorylate IKKα and IKKβ. The large(~800 kDa) IKK complex contains at least anIKK-α–β heterodimer bound to thenoncatalytic IKKγ subunits. IKKγ is thought tosequester further upstream activatingmolecules. The IKK complex phosphorylatesIκBα, IκBβ and IκBε at amino-terminalserines and p105 at carboxy-terminal serinesin the signal response domains (SRDs) ofthese proteins. p105 is depicted as abipartite molecule which sequesters, with itscarboxy-terminal half, other NF-κB subunits(p50 and Rel). The phosphorylated inhibitorsare degraded by the ubiquitin–proteasomepathway to release NF-κB p50–Rel dimers.Intracellular molecules whose essentialfunction for NF-κB activation is provengenetically are highlighted by a dark ring.

α

γ γ

TNF-R1

IL-1R

TRAF-6

IKK complex(es)

SRDSRD IκBαp105

p50

p65

p50

NIK

Cot

TAK1

TCR–CD3

MAP3Klayer

Receptor-inducedcomplexes

Plasmamembrane

TRAF-2 RIP

TAB1

p65

p50p50

Rel

Ubiquitin-conjugation

Proteasome

LPS

CD28

IRAK

Nuclear translocation

Gene regulation

CD14/TLR

TRADD MyD88

IL-18R

MEKK1

β

NF-κB

ECSIT


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Although the NF-κB p50/p52 double mutant mice are alsoosteopetrotic [25], their defect lies rather in the differentia-tion of hematopoietic precursors into mature osteoclasts.

Activation of the IKK complex: mediatorsconnecting receptor complexes to IKKs Activation of the IKK complex proceeds initially with thephosphorylation of IKKβ at two sites (Ser177 and Ser181) ofits activation (T) loop ([26•] and below). Several mitogen-activated protein kinase kinase kinases (MAP3Ks) havebeen suggested to be inducible IKK kinases (Figure 2).Overexpression of either NIK, MEKK1 (MAPK/ERK[extracellular-signal-regulated kinase] kinase kinase 1),MEKK2 or MEKK3 [27–29] leads to activation of IKKs andNF-κB reporter genes. Kinase inactive versions of IKKα orIKKβ inhibit NF-κB activation by MAP3Ks, implying thatthese kinases act upstream of the IKK complex. For NIK,direct activation of IKKα through phosphorylation of Ser176in the T loop has been shown [30]. Similarly, MEKK1 phos-phorylates IKKα in vitro [27]. The physiological relevanceof both findings remains unclear, as IKKα is not essential formediating cytokine signaling to NF-κB (see above).Although NIK preferentially activates IKKα and MEKK1favors JNK in transfected cells [31], each kinase can alsoactivate the other pathway. These data indicate that overex-pression of signaling proteins in such a tightly regulatedsystem might not reflect the physiological situation. Evenoverexpression of dominant-negative kinase mutants couldlead to cross-inhibition of different signaling pathways,either by replacing highly related kinases or by blockingaccess of IKKs to other potential upstream activators.

Other MAP3K-like kinases are thought to connect NIKand MEKK1 to the receptor complexes. Transforminggrowth factor β activated kinase 1 (TAK1), together withits activator TAK1-binding protein 1 (TAB1), may link

TRAF-6 to the NIK/IKK cascade to mediate NF-κB acti-vation by IL-1 [32]. Tumor progression locus 2 (TPL-2;also known as Cot) was proposed to mediate signals fromTCR–CD3 and CD28, activating NIK and thus inducingNF-κB [33]. But, because Cot activates several signalingpathways, including ERK, JNK, NF-AT (nuclear factor ofactivated T cells) and NF-κB, experiments with overex-pressed Cot mutants should be interpreted with caution.

Until very recently, it was not known how MEKK1 mightbe connected to receptor complexes. Now the adapterprotein ECSIT (evolutionarily conserved signaling inter-mediate in Toll pathways) has been identified, whichbridges TRAF-6 to MEKK1 and is specific for theToll/IL-1 pathways [34]. ECSIT promotes the processingof MEKK1 to an 80 kDa form, which correlates with NF-κB activation; moreover, transfection of a DrosophilaECSIT homolog into insect cells induced the productionof two antibacterial peptides, defensin and attacin, indicat-ing that ECSIT plays a role in innate immune function.

Conclusions made about the potential IKK-activating kinas-es and their upstream adaptors were mostly derived fromoverexpression studies of wild-type or dominant-negativemutants. In order to identify the physiological activator(s) ofthe IKK complex, however, gene knockout experiments willneed to be performed. It will be interesting to see whetherMAP3Ks are in fact redundant and whether different NF-κBactivating pathways converge directly at the IKK complex orfurther upstream. Perhaps different NF-κB inducers activatedistinct IKK kinases that subsequently activate the IKKcomplex, using NEMO/IKKγ as a docking surface.

A mechanism of how prolonged activation of the inflamma-tory response is prevented has been proposed [26•],whereby IKKβ, after the initial phosphorylation of its


Figure 3

IκBα

NF-κB

P

PIκBα

NF-κB

P

P

F-box

WDβTrCP

Skp1

Cul1

E1

Ub Ub Ub Ub

UbUb

NF-κBIκBα

NF-κBHOS

ROC1

IKK26S proteasome

E2

IκBα destruction


Induced IκBα degradation by the ubiquitin (Ub)–proteasome pathway.After phosphorylation by the activated IKK-complex at Ser32 andSer36, IκBα is recognized by a ubiquitin ligase complex. This containsas a scaffold component Cul1, which is bound to ROC1 and Skp1.Skp1 in turn is attached to the F-box of one of two highly-relatedsubstrate recognition components, β-TrCP or HOS. These F-box

proteins bind to the phosphorylated DSGψXS motif in IκBα, which isalso conserved in IκBβ and IκBε, through a conserved WD-40 repeatdomain. A ubiquitin-conjugating enzyme (E2) is recruited by Cul1 totransfer activated ubiquitin groups provided by ubiquitin-activatingenzyme (E1) to Lys21 and Lys22 of IκBα. The 26S proteasome rapidlydegrades ubiquitinated IκBβ, thereby releasing NF-κB.

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T loop, undergoes progressive autophosphorylation at a car-boxy-terminal serine cluster, resulting in inhibition of itscatalytic activity. Only T loop mutations in IKKβ, but not inIKKα, prevent cytokine- or NIK-triggered IKK activation.Thus, IKKα is not required for stimulation of IKK activity,but it may contribute to total IKK activity when complexedwith IKKβ, presumably by being activated by IKKβ [26•].In agreement with the studies on IKK-deficient mice, thesedata provide strong evidence that IKKβ is the major targetfor proinflammatory cytokine signaling.

Targets of the IKK complexThe IκBs, once phosphorylated by the IKK complex, aremarked for degradation and subsequent ubiquitination.The ubiquitin ligase complex that recognizes phosphory-lated IκBs has recently been revealed (Figure 3). Thecomplex consists of an F-box/WD-domain protein, β-TrCP(β-transducin-repeat-containing protein)/Slimb [35•–37•]or its homolog HOS (homologous to Slimb) [38], Skp1,Cdc53/Cul1 (Cullin) and ROC1 [39]. The recognition ele-ment detected by β-TrCP/Slimb in IκBα is DS*GLDS*,in the single letter code for amino acids where S* repre-sents serines 32 and 36 phosphorylated by IKKs.Interestingly, a similar sequence is found in β-catenin/Armadillo, a component of the Wnt/Wg signaling pathway,as well as in the HIV-1-encoded Vpu protein (for reviewssee [40,41]). β-catenin and the Vpu-interacting HIV recep-tor CD4 are also degraded by the ubiquitin-proteasomesystem, although they are phosphorylated by differentkinases. Thus, it appears that the consensus DSGψXSmotif (in which ψ denotes a hydrophobic amino acidresidue) is not sufficient for specific substrate recognitionby the IKKs.

The precursor of p50, p105, is phosphorylated by IKKα andIKKβ upon TNFα stimulation and degraded, leading tothe release of sequestered NF-κB subunits, including p50[42]. The major phosphorylation sites in p105 are three ser-ines in its carboxyl terminus, mutation of which stronglyreduced both basal and TNFα-induced phosphorylation ofp105 by IKKα and its subsequent degradation. These ser-ines are part of two elements related to the DSGψXS motif.

Belich et al. [43] have shown that Cot physically associateswith p105 and induces increased phosphorylation and pro-teolysis of p105; however, no direct phosphorylation of p105by Cot could be demonstrated. Perhaps, as for IκBα, Cotacts through the IKK complex to induce p105 phosphoryla-tion. The unique role of p105 as an IKK substrate inphysiological NF-κB responses has to be determined.

Conclusions and perspectivesThe pathways triggered by bacterial pathogens as part ofthe innate immune response to activate NF-κB convergewith those induced by cytokines at the level of intracellularsignal mediators and large multicomponent receptor andkinase complexes. In the past year analyses of receptor-associated signal mediators and of mice deficient in IKKs

have delineated further steps in the NF-κB activation path-ways, as well as revealing novel functions of IKK–NF-κB inskin differentiation. One major issue for future research isto identify the developmental stimuli that activate IKKαand to characterize NF-κB target genes in early skin mor-phogenesis. It is also not well understood how the differentreceptors are linked to the IKK complex by mediator mol-ecules and to what extent these are inducer-type specific orredundant for several pathways. Finally, each of the NF-κBmembers is known from knockout studies to have distinctfunctions, yet it is still entirely unknown how signalingpathways acting through a common IKK complex can dis-criminate between the various NF-κB heteromers toinitiate specific gene activation programs.

UpdateAfter completion of this review, further papers directly rel-evant to the topic were published.

Pomeranz and Baltimore [44] reported the identification ofTBK1, a novel kinase related to IKKα or β and 48% identi-cal to IKK-i, that associates with the TRAF-binding proteinTANK (I-TRAF). TBK1 mediates NF-κB activation byTRAF-2, -5, -6 and TANK. TBK1 forms a complex withTANK and TRAF-2 that functions upstream of NIK and theIKK complex, but TBK1 does not appear to be required forthe activation of NF-κB by TNFα, IL-1 or CD40. Thus,TANK and TBK1 probably belong to a separate pathwayinduced by different stimuli. Since these data were obtainedentirely from transfection studies, the physiological impor-tance of TBK1 for NF-κB activation needs to be established.

Another recent paper [45] studied TRAF-2 deficient micein greater detail, following an earlier report [46] that showednormal activation of NF-κB in response to TNFα in thesemice. This extended analysis concluded that CD40-inducedproliferation and NF-κB activation in mutant splenocyteswere completely abrogated. Therefore, TRAF-2 andTRAF-6 (see above) are essential for CD40 signaling.Unexpectedly, TRAF-2 deficient macrophages producedincreased amounts of nitric oxide and TNFα after TNFαstimulation. The mechanism for TRAF-2-mediated nega-tive regulation of TNF signals is unknown at present.

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NF-κB and the innate immune response