NF-κB inhibitor targeted to activated endothelium ...travasation of inflammatory cells in...

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NF-κB inhibitor targeted to activated endothelium demonstrates a critical role of endothelial NF-κB in immune-mediated diseases Bettina Sehnert a,1 , Harald Burkhardt b,1 , Johannes T. Wessels c , Agnes Schröder d , Michael J. May e , Dietmar Vestweber f , Jochen Zwerina g , Klaus Warnatz a , Falk Nimmerjahn h , Georg Schett g , Stefan Dübel i , and Reinhard Edmund Voll a,2 a Department of Rheumatology and Clinical Immunology and Centre of Chronic Immunodeciency, University Medical Centre and University of Freiburg, 79106 Freiburg, Germany; b Division of Rheumatology, Department of Internal Medicine II and Fraunhofer IME-Project-Group Translational Medicine and Pharmacology, Johann Wolfgang Goethe University Frankfurt am Main, 60590 Frankfurt am Main, Germany; c Central Core Facility Molecular and Optical Live Cell Imaging, Department of Nephrology and Rheumatology, University Medicine, 37075 Göttingen, Germany; d Interdisciplinary Center of Clinical Research, Research Group N2, Nikolaus Fiebiger Center of Molecular Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany; e School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; f Max-Planck Institute for Molecular Biomedicine, 48149 Münster, Germany; g Department of Internal Medicine 3, University of Erlangen-Nürnberg, 91054 Erlangen, Germany; h Division of Genetics, Department of Biology, University of Erlangen-Nürnberg, 91058 Erlangen, Germany; and i Institute of Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, 38106 Braunschweig, Germany Edited by Florian R. Greten, Technical University of Munich, Munich, Germany, and accepted by the Editorial Board August 2, 2013 (received for review October 18, 2012) Activation of the nuclear transcription factor κB (NF-κB) regulates the expression of inammatory genes crucially involved in the pathogenesis of inammatory diseases. NF-κB governs the expres- sion of adhesion molecules that play a pivotal role in leukocyteendothelium interactions. We uncovered the crucial role of NF-κB activation within endothelial cells in models of immune-mediated diseases using a sneaking ligand construct(SLC) selectively inhibiting NF-κB in the activated endothelium. The recombinant SLC1 consists of three modules: (i ) an E-selectin targeting domain, (ii )a Pseudomonas exotoxin A translocation domain, and (iii ) a NF-κB Essential Modier-binding effector domain interfering with NF-κB activation. The E-selectinspecic SLC1 inhibited NF- κB by interfering with endothelial IκB kinase 2 activity in vitro and in vivo. In murine experimental peritonitis, the application of SLC1 drastically reduced the extravasation of inammatory cells. Furthermore, SLC1 treatment signicantly ameliorated the disease course in murine models of rheumatoid arthritis. Our data establish that endothelial NF-κB activation is critically involved in the patho- genesis of arthritis and can be selectively inhibited in a cell type- and activation stage-dependent manner by the SLC approach. Moreover, our strategy is applicable to delineating other pathogenic signaling pathways in a cell type-specic manner and enables selective target- ing of distinct cell populations to improve effectiveness and riskbenet ratios of therapeutic interventions. cell targeting | intracellular signaling | autoimmune disorders | mouse models | inhibit inammation A critical step in the effector phase of pathogenic immune responses is the extravasation of circulating leukocytes from the vasculature and their migration along gradients of chemo- attractants into the target tissues (1). In inammation, endothelial activation represents a multistep cascade of events leading to in- creased vascular permeability for plasma proteins; the expression of proinammatory cytokines, chemokines, and enzymes; and an up-regulation of adhesion molecules that are regulated by the nuclear transcription factor κB (NF-κB) (24). Activation of NF-κB via the IκB kinase (IKK) complex is regarded as the classical NF-κB pathway. The IKK complex contains the kinases IKK1 and IKK2 and the regulatory subunit NF-κB Essential Modier (NEMO) (5). Activation of the clas- sical NF-κB pathway requires association of NEMO with IKK2 (5). The expression of multiple proinammatory genes including the adhesion molecules ICAM-1, VCAM-1, E-selectin, and che- mokines, like MCP-1 and IL-8, contributes to the inammatory endothelial cell response and is initiated through activation of the classical NF-κB pathway (6). Deregulated NF-κB has been implicated in the pathogenesis of immune-mediated inammatory diseases such as rheumatoid arthritis (RA) (79). In animal models, arthritis could be ame- liorated by general NF-κB inhibition (10, 11). However, NF-κB activation is also involved in the resolution of inammation and hence may exert positive or negative effects on inammatory processes depending on the cell type and the disease phase (12). Thus far, attempts to interfere with endothelial cell function as a therapeutic strategy in arthritis have remained rather limited and focused on the inhibition of synovial neoangiogenesis (13) or on attempts to directly block leukocyte extravasation by mono- clonal antibodies targeting selectins and/or endothelial integrins in different experimental arthritis models (3, 10, 1417) and patients (18). However, it is still poorly understood whether the extent of tissue damage in immune-mediated inammatory dis- eases like RA is directly dependent on enhanced leukocyte re- cruitment in response to NF-κB activation and whether the endothelium is the critical compartment of NF-κB activity. Filling Signicance The transcription factor NF-κB is crucially involved in the pathogenesis of inammatory diseases and represents a target for treatment. However, a general blockade of NF-κB by small- molecule inhibitors is associated with serious side effects due to the importance of NF-κB in cellular survival and function of various organs. This paper demonstrates that cell type-specic NF-κB inhibition can be achieved using multi-modular fusion proteins, which exclusively target activated endothelial cells. Inhibition of NF-κB within activated endothelium potently ameliorated peritonitis and arthritis in mice, indicating that endothelial NF-κB might be a valid target in inammatory diseases. Importantly, this strategy enables the targeting of other cell types and intracellular signaling pathways. Author contributions: B.S., H.B., S.D., and R.E.V. designed research; B.S., J.T.W., A.S., K.W., and R.E.V. performed research; M.J.M. and D.V. contributed new reagents/analytic tools; B.S., H.B., J.T.W., D.V., J.Z., F.N., G.S., and R.E.V. analyzed data; F.N. provided G6PI serum; and B.S., H.B., M.J.M., and R.E.V. wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission. F.R.G. is a guest editor invited by the Editorial Board. Freely available online through the PNAS open access option. 1 B.S. and H.B. contributed equally to this work. 2 To whom correspondence should be addressed. E-mail: [email protected] de. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1218219110/-/DCSupplemental. 1655616561 | PNAS | October 8, 2013 | vol. 110 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1218219110

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  • NF-κB inhibitor targeted to activated endotheliumdemonstrates a critical role of endothelial NF-κBin immune-mediated diseasesBettina Sehnerta,1, Harald Burkhardtb,1, Johannes T. Wesselsc, Agnes Schröderd, Michael J. Maye, Dietmar Vestweberf,Jochen Zwerinag, Klaus Warnatza, Falk Nimmerjahnh, Georg Schettg, Stefan Dübeli, and Reinhard Edmund Volla,2

    aDepartment of Rheumatology and Clinical Immunology and Centre of Chronic Immunodeficiency, University Medical Centre and University of Freiburg,79106 Freiburg, Germany; bDivision of Rheumatology, Department of Internal Medicine II and Fraunhofer IME-Project-Group Translational Medicine andPharmacology, Johann Wolfgang Goethe University Frankfurt am Main, 60590 Frankfurt am Main, Germany; cCentral Core Facility “Molecular and OpticalLive Cell Imaging”, Department of Nephrology and Rheumatology, University Medicine, 37075 Göttingen, Germany; dInterdisciplinary Center of ClinicalResearch, Research Group N2, Nikolaus Fiebiger Center of Molecular Medicine, University of Erlangen-Nürnberg, 91054 Erlangen, Germany; eSchool ofVeterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; fMax-Planck Institute for Molecular Biomedicine, 48149 Münster, Germany;gDepartment of Internal Medicine 3, University of Erlangen-Nürnberg, 91054 Erlangen, Germany; hDivision of Genetics, Department of Biology, University ofErlangen-Nürnberg, 91058 Erlangen, Germany; and iInstitute of Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, 38106Braunschweig, Germany

    Edited by Florian R. Greten, Technical University of Munich, Munich, Germany, and accepted by the Editorial Board August 2, 2013 (received for reviewOctober 18, 2012)

    Activation of the nuclear transcription factor κB (NF-κB) regulatesthe expression of inflammatory genes crucially involved in thepathogenesis of inflammatory diseases. NF-κB governs the expres-sion of adhesion molecules that play a pivotal role in leukocyte–endothelium interactions. We uncovered the crucial role of NF-κBactivation within endothelial cells in models of immune-mediateddiseases using a “sneaking ligand construct” (SLC) selectivelyinhibiting NF-κB in the activated endothelium. The recombinantSLC1 consists of three modules: (i) an E-selectin targeting domain,(ii ) a Pseudomonas exotoxin A translocation domain, and (iii)a NF-κB Essential Modifier-binding effector domain interferingwith NF-κB activation. The E-selectin–specific SLC1 inhibited NF-κB by interfering with endothelial IκB kinase 2 activity in vitroand in vivo. In murine experimental peritonitis, the application ofSLC1 drastically reduced the extravasation of inflammatory cells.Furthermore, SLC1 treatment significantly ameliorated the diseasecourse in murine models of rheumatoid arthritis. Our data establishthat endothelial NF-κB activation is critically involved in the patho-genesis of arthritis and can be selectively inhibited in a cell type- andactivation stage-dependent manner by the SLC approach. Moreover,our strategy is applicable to delineating other pathogenic signalingpathways in a cell type-specific manner and enables selective target-ing of distinct cell populations to improve effectiveness and risk–benefit ratios of therapeutic interventions.

    cell targeting | intracellular signaling | autoimmune disorders |mouse models | inhibit inflammation

    Acritical step in the effector phase of pathogenic immuneresponses is the extravasation of circulating leukocytes fromthe vasculature and their migration along gradients of chemo-attractants into the target tissues (1). In inflammation, endothelialactivation represents a multistep cascade of events leading to in-creased vascular permeability for plasma proteins; the expressionof proinflammatory cytokines, chemokines, and enzymes; and anup-regulation of adhesion molecules that are regulated by thenuclear transcription factor κB (NF-κB) (2–4).Activation of NF-κB via the IκB kinase (IKK) complex is

    regarded as the classical NF-κB pathway. The IKK complexcontains the kinases IKK1 and IKK2 and the regulatory subunitNF-κB Essential Modifier (NEMO) (5). Activation of the clas-sical NF-κB pathway requires association of NEMO with IKK2(5). The expression of multiple proinflammatory genes includingthe adhesion molecules ICAM-1, VCAM-1, E-selectin, and che-mokines, like MCP-1 and IL-8, contributes to the inflammatoryendothelial cell response and is initiated through activation of theclassical NF-κB pathway (6).

    Deregulated NF-κB has been implicated in the pathogenesisof immune-mediated inflammatory diseases such as rheumatoidarthritis (RA) (7–9). In animal models, arthritis could be ame-liorated by general NF-κB inhibition (10, 11). However, NF-κBactivation is also involved in the resolution of inflammation andhence may exert positive or negative effects on inflammatoryprocesses depending on the cell type and the disease phase (12).Thus far, attempts to interfere with endothelial cell function as

    a therapeutic strategy in arthritis have remained rather limitedand focused on the inhibition of synovial neoangiogenesis (13) oron attempts to directly block leukocyte extravasation by mono-clonal antibodies targeting selectins and/or endothelial integrinsin different experimental arthritis models (3, 10, 14–17) andpatients (18). However, it is still poorly understood whether theextent of tissue damage in immune-mediated inflammatory dis-eases like RA is directly dependent on enhanced leukocyte re-cruitment in response to NF-κB activation and whether theendothelium is the critical compartment of NF-κB activity. Filling

    Significance

    The transcription factor NF-κB is crucially involved in thepathogenesis of inflammatory diseases and represents a targetfor treatment. However, a general blockade of NF-κB by small-molecule inhibitors is associated with serious side effects dueto the importance of NF-κB in cellular survival and function ofvarious organs. This paper demonstrates that cell type-specificNF-κB inhibition can be achieved using multi-modular fusionproteins, which exclusively target activated endothelial cells.Inhibition of NF-κB within activated endothelium potentlyameliorated peritonitis and arthritis in mice, indicating thatendothelial NF-κB might be a valid target in inflammatorydiseases. Importantly, this strategy enables the targeting ofother cell types and intracellular signaling pathways.

    Author contributions: B.S., H.B., S.D., and R.E.V. designed research; B.S., J.T.W., A.S., K.W.,and R.E.V. performed research; M.J.M. and D.V. contributed new reagents/analytic tools;B.S., H.B., J.T.W., D.V., J.Z., F.N., G.S., and R.E.V. analyzed data; F.N. provided G6PI serum;and B.S., H.B., M.J.M., and R.E.V. wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS Direct Submission. F.R.G. is a guest editor invited by theEditorial Board.

    Freely available online through the PNAS open access option.1B.S. and H.B. contributed equally to this work.2To whom correspondence should be addressed. E-mail: [email protected]

    This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental.

    16556–16561 | PNAS | October 8, 2013 | vol. 110 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1218219110

    mailto:[email protected]:[email protected]://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplementalwww.pnas.org/cgi/doi/10.1073/pnas.1218219110

  • this gap in our knowledge not only will improve our understandingof the pathogenic mechanisms but also may help to develop tar-geted therapeutic strategies. Moreover, NF-κΒ activation in thevascular endothelium is crucially involved in the development ofatherosclerosis. The transgenic expression of an IκΒα mutant inendothelial cells protected Apo E−/− mice (Tie2DNIkBa/ApoE−/−mice) from atherosclerosis (19), and myeloid-specific IKKβ de-letion decreased atherosclerosis in LDL receptor-deficient mice(20). Repetitive systemic inflammatory responses are character-istic features of autoimmune diseases like RA that compose acytokine-mediated activation of the endothelium. Accordingly,evaluation of the pathogenicity of endothelial NF-κΒ might iden-tify treatment options for inflammatory tissue damage as well asits accompanying systemic comorbid conditions (21).In this study, we investigated the role of NF-κB in the acti-

    vated endothelium in murine models of peritonitis and RA. Wedeveloped an E-selectin–specific NF-κB inhibitor to selectivelytarget activated endothelial cells. This fusion protein, designatedas “sneaking-ligand construct” (SLC) 1, consists of a ligand-binding motif for high avidity interaction with E-selectin andendocytosis (22), a translocation domain to ensure endosomalrelease (23) and a NEMO-binding peptide (NBP) disrupting theIKK complex (24). This approach to cell type-specific and acti-vation status-dependent targeting of NF-κB revealed that the ex-travasation of inflammatory cells in peritonitis as well as synovialleukocyte infiltration in arthritis is critically dependent on endo-thelial NF-κB activation via the classical pathway. Thus, we providea proof of concept for the therapeutic application of an endothelialcell-specific NF-κB blockade in immune-mediated diseases.

    ResultsGeneration of Sneaking Ligands Inhibiting NF-κB in the ActivatedEndothelium. Transgenic and knockout mice have been used toclarify the role of molecules and signaling pathways in endo-thelial cells for the pathogenesis of diseases (19, 25). However,this approach is time-consuming and costly and does not allowthe investigation of the effects of short-term modulation unlesscomplex inducible systems are used. To gain insight into thefunction of NF-κB in activated endothelium, we took advantageof cytokine-induced E-selectin expression (2–4) and engineeredthe modular NF-κB inhibitor SLC1 for bacterial expression (26).The prototypic SLC1 is composed of three modules: (i) thetargeting domain, consisting of three repeats of an E-selectin–binding peptide (AF10166 DITWDQLWDLMK) termed EBL(22) that confers high avidity to E-selectin and leads to receptor-mediated endocytosis, (ii) the translocation domain of Pseudo-monas exotoxin A (ETAII) (23) to facilitate endosomal releaseof the effector domain into the cytosol, and (iii) the NBPencompassing amino acids 644–756 of IKK2 to serve as the ef-fector domain for inhibition of the classical NF-κB pathway (24,27). The integrated KDEL retention signal supports the trans-port of SLCs to the ER in a retrograde manner and the sub-sequent release into the cytsol (28). In Fig. 1, the composition ofSLC1 is illustrated and its amino acid sequence is shown in Fig.S1A. Fig. 1 also depicts additional fusion proteins generated tocontrol for the functional contributions of the EBL domain[mutated to a nonfunctional variant (MutEBL) or deletion var-iant (DelEBL)], the NBP domain [mutated to nonfunctionalvariants in MutNBP1 and MutNBP2], and the ETAII domain[DelETA, deletion of ETAII domain]. SDS/PAGE (Fig. S1B)and Western blot (Fig. S1C) analyses demonstrated high puritiesof the recombinant proteins. Using an antibody against the N-terminal Strep-Tag, we identified SLC1 as well as MutEBL,MutNBP1, and MutNBP2 migrating at the expected molecularweight of 39 kDa. The control construct DelEBL has a size of 32kDa (Fig. S1C). An IKK2-specific antibody was applied for thedetection of the C-terminal NBP domain to prove the expressionof full-length proteins (Fig. S1D).First, we tested the binding capacity of our fusion protein by

    cell-based ELISA. SLC1 and MutNBP2 bound specifically to cellstransfected with mouse E-selectin (CHO_E), whereas MutEBL

    and DelEBL did not, indicating the importance of the functionalEBL domain for E-selectin binding (Fig. S1E). Furthermore,SLC1 did not interact with ICAM-1–transfected CHO cells(CHO_I) or wild-type CHO cells (CHO_wt), confirming itsspecificity for E-selectin (Fig. S1F).

    NF-κB Inhibition via E-Selectin–Mediated Endocytosis of SLC1 in Vitro.The translocation of surface-bound SLC1 into the cytoplasm isa prerequisite for inhibition of NF-κB activation. For thesestudies, the infrared-fluorescent protein FP635 (mKATE) wascloned into the sneaking ligand constructs for their visualization(29, 30). We examined the uptake of SLC1-FP635 into CHO_Ecells by immunofluorescence microscopy. A temperature-sensi-tive receptor-mediated endocytotic uptake was indicated by theintracellular distribution of SLC1-FP635 upon incubation at 37 °Cfor 90 min (Fig. 2A), whereas parallel experiments performed at4 °C resulted in a SLC1-FP635 staining pattern that colocalizedwith the fluorescence signal of the AlexaFluor488-labeled ConAplasma membrane marker (Fig. 2B).We have previously established that an antennapedia-linked

    cell-permeable NBP (Antp-NBP) blocks the association ofNEMO with IKK2 and inhibits activation of the classical NF-κBpathway (24). To analyze the effects of SLC1 treatment on cy-tokine-induced NF-κB activity, we performed NF-κB–dependentluciferase reporter gene assays in the CHO cell lines and com-pared the effects of SLC1 with Antp-NBP. CHO_E or CHO_wtcells were pretreated for 90 min at 37 °C with a range of con-centrations of SLC1 (125–500 nM), then NF-κB was activated byIL-1β. Transcriptional NF-κB activity was significantly reduced inSLC1-treated CHO_E cells (Fig. 2C), but not in CHO_wt cells(Fig. 2D). In contrast, Antp-NBP reduced the transcriptionalNF-κB activity in both CHO_E and CHO wt cells, whereas themutated Antp-NBP (Antp-MutNBP) did not affect NF-κB ac-tivity (Fig. 2 C and D). Consistent with the previous finding thatcell-permeable NBP treatment does not block basal NF-κB ac-tivity (24), SLC1 did not decrease NF-κB activity in resting cellsbut prevented the cytokine-mediated NF-κB induction (Fig. 2C).We did not detect significant effects on the transcriptional NF-κBactivity in cells treated with the controls MutEBL, DelEBL,MutNBP1, and MutNBP2.We next measured the NF-κB DNA-binding activity of nuclear

    extracts from SLC1-treated cells using electrophoretic mobilityshift assays (EMSA). Incubation of CHO_E cells with SLC1 be-fore incubation with IL-1β resulted in a reduced NF-κB DNA-binding activity, indicating that SLC1 inhibits the IKK complexactivity and, thereby, prevents the translocation of NF-κB proteinsinto the nucleus (Fig. 2E). In contrast, MutNBP2, MutEBL, andDelETA treatment did not affect NF-κB activation (Fig. 2E).Moreover, SLC1 treatment (500 nM) decreased DNA-bindingactivity to a similar level as Antp-NBP (10 μM). Quantitativedensitometry of independent EMSA experiments revealed a sig-nificant reduction in NF-κB DNA-binding activity in SLC1-treatedCHO_E cells (67.5% compared with IL-1β control) (Fig. 2E).EMSA supershift analyses showed that the NF-κB complexes inIL-1β–activated cells consisted almost exclusively of p50/p65complexes, indicating activation of the classical NF-κB pathway. InSLC1-treated activated cells, the NF-κB DNA-binding activity wasmarkedly reduced, but still virtually all complexes were shiftedwith antibodies to p50 and p65 (Fig. S2A). Furthermore, SLC1 didnot affect p38 or AP-1 activation (c-Jun, JunB) in cytokine-acti-vated CHO_E cells (Fig. S2B). Collectively, these data establishthat the EBL and NBP domains of SLC1, as well as the ETAIIdomain, are essential for NF-κB inhibition and that SLC1 selec-tively targets the classical NF-κB pathway.

    Endothelial NF-κB Blockade with SLC1 Prevents Leukocyte Trafficking.The interaction of leukocytes with endothelium is mediated byNF-κB–dependent adhesion molecules expressed on activatedendothelial cells. We therefore questioned whether interferencewith the IKK complex assembly significantly influenced leukocytebinding to and migration across cytokine-activated endothelium.

    Sehnert et al. PNAS | October 8, 2013 | vol. 110 | no. 41 | 16557

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    http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF2

  • IL-1β−stimulated mouse endothelial cells (mlEND) were incu-bated for 4 h with SLC1 before adding granulocytes. Pretreatmentof endothelial cells with SLC1 but not with the dysfunctionalcontrols MutNBP2, MutEBL, and DelEBL caused a reduction inthe percentage of adherent granulocytes (Fig. 3A). Incubation ofcytokine-activatedmlEND cells with Antp-NBP also decreased thenumber of adherent granulocytes. Accordingly, NF-κB inhibitionby both SLC1 treatment and Antp-NBP caused impaired trans-migration of granulocytes across the activated endothelial celllayer (Fig. 3B).To investigate whether a NF-κB blockade in endothelial cells

    alters leukocyte trafficking in vivo, we used a mouse model ofacute thioglycollate-induced peritonitis. As shown in Fig. 3C,leukocyte influx into the peritoneal cavity of SLC1-treated micewas strongly reduced whereas equimolar doses of MutNBP2 andMutEBL did not exert significant effects. Hence, these data in-dicate that SLC1 inhibits inflammatory cell recruitment byblocking NF-κB in activated endothelial cells in vivo.Because a leaky vasculature is a characteristic feature of in-

    flammatory disorders (31), we also studied the modulatory poten-tial of SLC1 on cytokine-induced increase of vascular permeabilityin vivo. As a surrogate for plasma extravasation, Evans blue wasquantified in extracts of skin biopsies obtained from cytokine-exposed areas. Blockade of IKK2 in SLC1-treated mice resultedin significantly reduced vascular permeability compared with un-treated, MutNBP2-, or MutEBL-treated mice (Fig. 3D).

    In Vivo Imaging of SLC1 Binding to Cytokine-Activated VascularEndothelium. In vivo imaging was used to prove the selectivebinding of SLC1 to cytokine-activated but not to resting endo-thelium in vivo (29, 30). An epigastric vein in a cytokine-inducedskin flap of live transgenic green fluorescent protein (GFP)-expressing mice was selected as the region of interest for SLC1detection (Fig. S3A). Intraperitoneal injections of the infrared-labeled SLC1-FP635 into GFP mice did not lead to any changeof the green fluorescent appearance of the nonactivated vascu-lature (Fig. 4A, Top). However, upon local cytokine challenge bythe simultaneous injection of IL-1β and TNF-α (Fig. 4A,Middle),SLC1-FP632 (red) was localized to the vessel wall, resulting in anintensely stained orange border at the endothelial lining, whichmost likely reflects specific binding of SLC1 to E-selectin andsubsequent internalization. Under identical conditions the sys-temically administered FP635-tagged DelEBL control constructdid not accumulate in the vessel wall as reflected by the un-changed green fluorescent appearance of the cytokine-activated

    endothelial lining (Fig. 4A, Bottom). The luminal red fluores-cence signal was detectable upon administration of FP635-labeledSLC1 and the FP635-DelEBL mutant under cytokine-activatedbut not under noninduced conditions (Fig. 4A and Fig. S3B). Thisobservation was consistent in all mice and is most likely explainedby reduced blood flow velocity and changes in the glycocalix ofactivated endothelial cells, promoting unspecific surface inter-actions with proteins. However, there was clearly no accumula-tion in the endothelial cell layer, as also shown by analysis of pixelintensities, which were calculated from distinct cross-sections atrandomly selected positions along the axes of independent ves-sels. The grayscale pixel values of SLC1-FP635– and DelEBL-FP635–treated mice significantly differed at the vessel wall areasand clearly demonstrate selective accumulation of SLC1 in theendothelial layer (Fig. S3C).

    Decreased NF-κB Activity in Cytokine-Activated Mouse Skin uponSLC1 Treatment. Endothelial NF-κB activation was investigatedex vivo by whole-mount immunofluorescence microscopy of tis-sue specimens from cytokine-activated mouse skin upon doublestaining with a monoclonal antibody selectively binding to theactivated, nuclear form of NF-κB p65 (32) and a VCAM-1–specific antibody. The adhesion molecule VCAM-1, which isstrongly expressed due to cytokine-induced NF-κB activation,was stained simultaneously to identify the vascular endothelium.In addition, the amount of VCAM-1 expression reflects thetranscriptional activity of NF-κB. Cytokine-induced nuclear NF-κB p65 staining in the vascular endothelium was strongly sup-pressed in mice pretreated with SLC1 but not in control mice that

    Fig. 1. Schematic representation of functional and nonfunctional SLCs. Themultimodular synthetic gene was ligated into the pAK400 plasmid usingSfiI restriction sites. EBL, three repeats of E-selectin–specific peptide(DITWDQLWDLMK) connected with a S4G linker. MutEBL, WKLDTLDMIQD(22). ETA II, translocation domain of Pseudomonas exotoxin A domain II (23).NBP, NEMO-binding peptide encompassing amino acids 644–756 from IKK2(24). Amino acids that were identified for NEMO interaction were mutated:MutNBP1 FTALDASALQTE. MutNBP2 (scrambled peptide): DLAWQTFLTES.

    Fig. 2. Internalized SLC1 selectively inhibits NF-κB activation in vitro. (A)Immunofluorescence microscopy depicts CHO_E cells stained with SLC1-FP635 at 37 °C. Plasma membrane was stained with AlexaFluor488-labeledConA. (B) CHO_E cells were stained with SLC1-FP635 at 4 °C. Experimentswere performed at least five times. (C) NF-κB–dependent luciferase expression isreduced in SLC1-pretreated CHO_E cells in a dose-dependent manner. Error barsrepresent the ±SD obtained from five independent experiments. Each experi-ment was done in triplicate. (D) Transcriptional NF-κB activity is not reduced bySLC1 in CHO_wt cells. (E) NF-κB EMSA in SLC1-pretreated and IL-1β–stimulatedCHO_E cells demonstrates inhibition of NF-κB nuclear DNA-binding activity.Representative independent experiments are shown. Experiments were per-formed five times. Densitometric analysis shows inhibition of NF-κB DNA-bindingactivity by ∼35% upon SLC1 treatment. Densitometric measurements were per-formed from five separate experiments. P values were calculated by one-wayANOVA followed by Bonferroni’s multiple-comparison post test: ****P < 0.0001;***P < 0.001, **P < 0.01, *P < 0.05.

    16558 | www.pnas.org/cgi/doi/10.1073/pnas.1218219110 Sehnert et al.

    http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF3http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF3http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218219110/-/DCSupplemental/pnas.201218219SI.pdf?targetid=nameddest=SF3www.pnas.org/cgi/doi/10.1073/pnas.1218219110

  • had received the MutNBP2 fusion protein (Fig. 4B). Accordingly,the skin of SLC1- but not of MutNBP2-pretreated mice exhibiteda reduced endothelial VCAM-1 expression (Fig. 4B). Only weakVCAM-1 expression and no nuclear p65 staining were observed innonactivated skin. These observations were confirmed by quan-tifying the mean fluorescence intensities of p65 and VCAM-1stainings (Fig. S3D). Thus, our data indicate that a systemicallyadministered NF-κB inhibitor targeting E-selectin suppresses en-dothelial NF-κB activation and concomitant VCAM-1 expressionin a cytokine-induced inflammatory response in vivo.

    NF-κB Inhibition in Endothelial Cells Ameliorates ExperimentalArthritis. Next, we asked whether NF-κB activation in endothe-lial cells is critically involved in the pathogenesis of serum transferarthritis (STIA) (33) and antigen-induced arthritis (AIA) (34). Inthe STIA model, injection of serum from K/BxN mice containingantibodies against glucose-6-phosphoisomerase (anti-G6PI) causesan acute polyarthritis. The aggressive STIA shares many featureswith human RA, including polyarticular manifestation, leukocyteinvasion, pannus formation, synovitis, as well as cartilage and boneerosions (33). Mice were intraperitoneally injected with the au-toantibody-containing serum and the SLC proteins (100 μg/in-jection). SLC application was repeated after 7 and 24 h. Under thesame conditions we examined the effect of Antp-NBP (200 μg/injection) in STIA. The clinical arthritis scores were significantlylower in SLC1-treated mice compared with PBS-, MutNBP2-, andDelEBL-treated mice. The therapeutic effect of SLC1 was com-parable to Antp-NBP (Fig. 5A).The extension of SLC1 or Antp-NBP treatments by additional

    applications at days 2, 4, and 5 did not result in more pro-nounced reduction of disease severity (Fig. 5B). These data arein line with SLC1 acting as an inhibitor of extravasation in theearly phase of inflammation. Joint swelling was assessed bymeasuring the diameters of paws and ankles as shown in Fig.

    5C. Swelling was significantly reduced in mice treated withSLC1 or Antp-NBP compared with the control groups. At theend of the experiment, hindpaws were analyzed by histology.SLC1 application resulted in suppressed cartilage and bonedegradation, as well as reduced synovial inflammation (Fig. 5D and E).AIA is induced by intra-articular injection of methylated BSA

    into the knee joints of mice immunized with methylated BSA,resulting in an acute monoarthritis lasting approximately 1 wk.During the chronic phase of AIA, intra-articular methylated BSAinjection causes a flare-up reaction of arthritis. Intraperitonealapplication of SLC1 resulted in reduced joint swelling during theacute (Fig. S4A) as well as flare-up phase (Fig. S4B) compared withcontrol mice treated with solvent, DelEBL, or MutNBP2. Histo-logical analyses supported the clinical findings. Joints of SLC1-treated mice exhibited the normal appearance of a thin synoviallining layer with only a sparse infiltration by inflammatory cells(indicated by the arrows) compared with the hyperplastic synoviumwith diffuse infiltrations of numerous leukocytes in PBS-treatedmice (Fig. S4 C and D). These results indicate that NF-κB acti-vation within endothelial cells is a crucial event in the patho-genesis of experimental models for RA.

    DiscussionA hallmark of inflammation in autoimmune disorders such asRA is the activation of the classical NF-κB pathway. Accordingly,targeting NF-κB for anti-inflammatory intervention has beensuccessfully demonstrated in multiple animal models during pastyears (10, 24, 35). However, systemic interference with NF-κBactivation influences a variety of homeostatic regulatory path-ways in, for example, the liver and the immune system (16).Ubiquitous pharmacologic suppression of NF-κB activity maytherefore lead to severe side effects including profound immu-nosuppression, liver cell apoptosis, and organ dysfunctions (16,17, 36). The SLC approach is likely to have an improved risk–benefit ratio compared with a general NF-κB inhibition (16, 17,36, 37). As NF-κB is also involved in the resolution of in-flammation, systemic inhibition of its activity can even promotechronic inflammation depending on the cell type and the diseasephase (12, 38). Moreover, IKK2 can exert direct anti-inflammatoryproperties via inhibition of classical macrophage activation andcounteraction of IL-1β secretion. Hence, IKK2 is critical to pre-vent IL-1β–driven neutrophilia (39). Therefore, a more efficientand safer therapy should target selectively those cell types in which

    Fig. 3. SLC1 reduces leukocyte–endothelium interactions in vitro and in vivoand prevents vascular leakage. (A) Granulocyte adhesion was reduced inSLC1-treated and cytokine-stimulated mouse endothelial cells (mlEND).Experiments were performed five times in triplicate. (B) In vitro trans-migration of leukocytes through cytokine-activated mlENDs is inhibited bySLC1 application as well as by Antp-NBP. Experiments were performed fivetimes in triplicate. (C) SLC1 application inhibited neutrophil migration in vivoin murine peritonitis. PBS, n = 9; SLC1, n = 10; MutNBP2, n = 5; MutEBL, n = 5.(D) Extravasation of Evans blue is prevented in cytokine-induced skin ves-sels in SLC1-treated mice. PBS, n = 8; SLC1, n = 10; MutNBP2, n = 5; MutEBL,n = 6. P values were calculated by one-way ANOVA followed by Bonfer-roni’s multiple-comparison posttest: ****P < 0.0001; ***P < 0.001, **P <0.01, *P < 0.05. Error bars represent the ±SD.

    Fig. 4. SLC1 binding to the vascular endothelium in vivo is dependent onits cytokine-activation status: inhibition of NF-κB activation. (A) Visualizationof vessels in a nonactivated state (Top) exhibits the GFP-background fluo-rescence but no staining by the systemically administered red-fluorescentSLC1-FP635. Upon cytokine activation, the red fluorescent SLC1-FP635 accu-mulates in the vessel wall, leading to an intense orange staining (arrow,Middle) that is not detectable when the deletion mutant DelEBL-FP635 isadministered (Bottom). Experiments were performed three times. (B) Rep-resentative images of whole-mount immunofluorescence skin sectionsstained for activated NF-κB p65 and VCAM-1. Experiments were performedthree times.

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  • NF-κB actually contributes to the pathogenesis of a certain dis-ease. In this regard, the vascular endothelium is a selective barrierbetween blood and tissue and represents a checkpoint for extrav-asation of plasma proteins and inflammatory cells, preventing orallowing transmigration of neutrophils, monocytes, and T and Blymphocytes into the sites of inflammation (3, 4).Toward a better understanding of the role of NF-κB in the

    activated endothelium in experimental models of inflammation,we developed a tool to manipulate NF-κB activity selectively inactivated endothelial cells. For targeting, E-selectin has beenchosen due to its NF-κB–dependent transcriptional up-regulationin the early phase of cytokine activation and its internalizationupon ligation (40, 41).“Sneaking ligand” refers to the contribution of the ETAII

    domain to the SLC1 function, i.e., to promote the translocationof the NEMO-binding effector domain from the endosomalcompartment into the cytosol to achieve NF-κB blockade (28).Single-chain variable fragment immunotoxins were successfullyused to selectively kill cancer cells, indicating that the endosomalrelease function of the ETAII domain is not compromised inchimeric proteins (42). The essential role of the translocationdomain in our sneaking-ligand principle is demonstrated by theinability of the DelETA to inhibit NF-κB activation. Our inves-tigations provide unequivocal evidence for the crucial role ofSLC1 interaction with surface E-selectin for targeted cellularuptake. Thus, mutants of SLC1 harboring an intact NEMO-binding domain but lacking the E-selectin–interacting modulefailed to block NF-κB in E-selectin–expressing CHO cells in vitro.Upon systemic application in mice the deletion mutant DelEBL,in contrast to the full-length SLC1, did not accumulate in theendothelial layer of a cytokine-activated vasculature.SLC1 interfered with leukocyte binding and neutrophil mi-

    gration in vitro and in vivo. The crucial involvement of NF-κBinhibition is supported by control experiments with Antp-NBPand MutEBL that render alternative explanations for the mech-anism of SLC1 action such as masking of E-selectins or theirdown-regulation unlikely.The anti-inflammatory effects of an NF-κB blockade in en-

    dothelial cells was shown in two different models of arthritis.Inhibition of IKK2 in endothelial cells efficiently controlledmonoarticular arthritis in AIA as well as polyarticular arthritis inSTIA. Remarkably, the effective molar concentration of SLC1 was20-fold less than the concentration of Antp-NBP. Histologicaldata confirmed the SLC1-dependent anti-inflammatory effectand demonstrated that endothelial specific inhibition of NF-κBprotected cartilage and bone against inflammation-induced struc-tural damage. These results define the critical role of endothelialNF-κB activation in arthritis and are consistent with experiments

    blocking endothelial adhesion molecules, which amelioratedcollagen-induced arthritis (3, 14). The essential role of the NBPeffector domain was confirmed with constructs that contain anintact E-selectin–binding domain together with a nonfunctionalNBP (MutNBP2). These mutants lost their ability to block NF-κB activation in vitro as well as in vivo and also lacked anti-inflammatory activity in AIA and STIA in vivo despite their intactE-selectin–binding domain.Changes in vascular permeability represent a common feature

    of inflammatory responses. We observed a decreased vascularleakage in SLC1-treated mice, demonstrating that NF-κB activa-tion via the classical pathway is critical for an increased vascularpermeability. Our results are in accordance with a study showingthe reduction of LPS-mediated vascular permeability in endo-thelial cells by interference with NF-κB activation using transgenicoverexpression of IκBα (43). In contrast, Ashida and colleaguesdescribed increased vascular permeability in endothelial-specificIKK2-deficient mice. Increased vascular permeability was due tokinase-independent effects of IKK2 (25). Therefore, the use ofSLC1 appears to be more specific than the investigation of IKK2-deficient cells in investigating NF-κB–dependent effects.In summary, our study establishes that NF-κB activation within

    endothelial cells is a crucial step in the pathogenesis of experi-mental models of RA. Disruption of the IKK complex in endo-thelial cells efficiently controls the extravasation of inflammatorycells in arthritis models and might represent a treatment strategyin RA. The potential immunogenicity of SLC1 may impede long-term treatments. To decrease immunogenicity, we are currentlydeveloping sneaking ligands predominantly based on endogenousproteins. Thus, we have developed a tool to study the role of in-tracellular signaling pathways in specific cell types contributing tothe pathogenesis of diseases. The present investigation reports thein vivo efficacy of a cell type-specific guided NF-κB inhibitionspecifically targeting the activated endothelium at sites of in-flammation and selectively blocking a crucial proinflammatoryintracellular signaling cascade. Thus, this approach is applicable asan anti-inflammatory strategy that maintains the essential ho-meostatic functions of NF-κB while selectively interfering withits activation in the inflamed endothelium. Considering the im-portance of endothelial NF-κB activation in pathophysiologicalconditions including septic shock (44), procoagulant states suchas disseminated intravascular coagulation (45), atherogenesis(46), or tumor neoangiogenesis (47), the therapeutic potentialwith SLC1 is likely much broader.The “sneaking ligand concept” is easily transferable to target

    other cell types and signaling pathways by the exchange of thebinding and effector domains. Hence, this study demonstrates

    Fig. 5. Clinical and histological manifestations are ame-liorated in the K/BxN serum transfer model after SLC1treatment. (A) Mice were three times treated with 100 μgSLCs or 200 μg Antp-NBP as indicated by arrows. PBS, n =19; SLC1, n = 13; MutNBP2, n = 9; DelEBL, n = 5; Antp-NBP,n = 5. (B) Treatments with the indicated SLCs or Antp-NBPwere continued on days 2, 4, and 5. SLC1, n = 10; MutNBP2,n = 10; DelEBL, n = 10; Antp-NBP, n = 10. (C ) On day 8diameters of paws (paw and ankle) were measured. (D)Representative microphotographs of H&E-stained sectionsat day 8 after K/BxN serum transfer. (E) Histological analysesof synovitis, cartilage, and bone erosions. P values were cal-culated by one-way ANOVA followed by Bonferroni’s multi-ple-comparison post test: ****P < 0.0001; ***P < 0.001, **P <0.01, *P < 0.05.

    16560 | www.pnas.org/cgi/doi/10.1073/pnas.1218219110 Sehnert et al.

    www.pnas.org/cgi/doi/10.1073/pnas.1218219110

  • a promising strategy to identify disease-relevant signaling path-ways in defined cell types.

    Materials and MethodsA detailed description of materials and methods is provided as SI Materialsand Methods.

    Cloning of Plasmids Expressing E-Selectin–Specific Sneaking Ligands. Nucleo-tide sequences corresponding to the E-selectin–specific peptide AF 10166 (22)or the scrambled peptide AF 11678 (22), the ETA II domain (amino acid 253–364) (23), and the NBP (amino acids 644–756 from IKK2) (24) and mutatedNBP (24) were connected via glycine–serine linkers. Sequences of thedesigned gene were codon optimized for E. coli expression, synthesized(Bio&Sell), and cloned into pAK400 (26) via SfiI. For in vivo imaging experi-ments as well as for confocal microscopy, an infrared-fluorescent tag FP635(mKATE) (30) was cloned behind the NBP domain.

    Mouse Models. K/BxN serum transfer arthritis (33), antigen-induced arthritis(34), or thioglycollate-induced peritonitis were induced as described andused to analyze the therapeutic efficacy of the SLCs or Antp-NBP.

    In Vitro Experiments. Neutrophil adhesion and transmigration through SLC1-treated and cytokine-stimulated mouse endothelial cells were performed.Inhibition of NF-κB activation was investigated using NF-κB luciferase re-porter assay and EMSA. Whole-mount immunofluorescence technique wasused to identify activated p65 and VCAM-1 in SLC-treated mice.

    ACKNOWLEDGMENTS. We thank Daniela Graef, Elvedina Nendel, EugeniaScheffler, Rita Rzepka, Olga Greb, and Ina Stumpf for excellent technicalassistance. mlEND cells were kindly provided by Dr. Ruppert Hallmann. Wethank Dr. Frank Emmerich for coining the name “sneaking ligand” andDr. Olaf Broders and Dr. Bernd Voedisch for their supportive work. We alsothank Winfried Wels. This research was supported by the German ResearchFoundation (SFB 643 project B3 and A8; FOR 832, project 7; SachbeihilfeDU337/3-2 and BU 584/4-1); BMBF 01EO0803 Grant to the Centre of ChronicImmunodeficiency; the Doktor-Robert Pfleger Stiftung; the Interdisciplin-ary Center of Clinical Research of the University Erlangen; the FP7 fund-ing of the European Union (Masterswitch); the German Federal Ministry ofEducation and Research ArthroMark (project 4, 01 EC 1009C); the FederalState of Hessen (LOEWE-Project: Fraunhofer IME-Project-Group Transla-tional Medicine and Pharmacology, Goethe University Frankfurt); and Na-tional Institutes of Health/National Heart, Lung, and Blood Institute GrantRO1HL096642.

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