Preparation and Characterization of Agonistic Monoclonal Antibodies Against Toll-Like Receptor 4-MD-2 Complex

Bahrun, Uleng; Kimoto, Masao; Tsukamoto, Hiroki; Tsuneyoshi, Naoko; Kohara, Jun; Fukudome, Kenji

doi:10.1089/hyb.2007.0523

Cited by 17 publications

(20 citation statements)

References 26 publications

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“…Zanoni et al (12) reported the requirement for CD14 in TLR4 endocytosis and the production of IFN-β by LPS. We compared internalization of TLR4 induced by LPS to that induced by UT12, a MAb directed against a TLR4/MD2 epitope that acts as a TLR4 agonist (13,17). In WT macrophages, both LPS and UT12 induced TLR4 internalization, whereas in CD14 −/− macrophages only UT12 induced TLR4 endocytosis (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…TLR4 endocytosis and TRIF-mediated signaling were induced by treatment of macrophages with UT12, a mouse antibody directed against an epitope formed by TLR4/MD2 interaction (13,14), and small synthetic TLR4 ligands (1Z105 and 1Z204) that bind to MD2 and signal through both MyD88-dependent and TRIFdependent pathways in the absence of CD14 (16). Although it is possible that the UT12 monoclonal antibody also activates internalization through FcγR-dependent uptake of UT12/TLR4/ MD2 immune complexes, UT12 is a mouse IgG3 that has high affinity for FcRn and very low affinity/no affinity toward FcγRI, FcγRIIB, FcγRIII, and FcγRIV (38,39).…”

Section: Cd14-independent Endocytosis Of Tlr4 In Macrophages Renderedmentioning

confidence: 99%

“…UT12 is a monoclonal antibody (MAb) with specificity for the mouse TLR4/MD2 complex and mediates LPS-like signaling (13). It has been shown that UT12 induces endotoxic shock-like symptoms in mice including augmentation of TNF-α and IL-6.…”

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confidence: 99%

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CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

Rajesh

Perkins

Ireland

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

125

104

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Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88-and TIRdomain-containing adapter-inducing IFN-β (TRIF)-mediated signaling pathways. Recently, Zanoni et al. [(2011) Cell 147(4):868-880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli-induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-β in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88-nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12-or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 "pocket," completely blocked LPS-and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14. T oll-like receptor 4 (TLR4) signaling plays a crucial role in host defense against Gram-negative bacteria by recognizing the outer membrane component, lipopolysaccharide (LPS) (1-3). TLR4 signaling is initiated by transfer of an LPS monomer from LPS binding protein (LBP) to cluster of differentiation 14 (CD14) (GPI-linked or soluble). In turn, CD14 transfers monomeric LPS to myeloid differentiation factor 2 (MD-2), a protein that associates noncovalently with TLR4 (4). Appropriate ligand binding to MD2 results in dimerization of two TLR4/MD2 complexes (4). TLR4 is unique in that it is the only TLR that activates both myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing IFN-β (TRIF)-dependent signaling pathways (5, 6). MyD88-mediated, TLR4 signaling occurs mainly at plasma membranes and involves IL-1R-associated kinases phosphorylation, association of TNF-receptor-associated factor 6, and downstream signaling that results in NF-κB activation and induction of proinflammatory mediators such as TNF-α and IL-6 (7). In contrast, TRIF-mediated signaling in response to LPS occurs at the endosomal membrane after internalization of the TLR4 that, in turn, activates IFN regulatory factor 3 (IRF3), resulting in production of IFN-β, IP-10, and other IRF-3-dependent genes, as well as delayed NF-κB activation (8). Recent studies have shown that the endocytosis of TLR4 is tight...

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Section: Resultsmentioning

confidence: 99%

Section: Cd14-independent Endocytosis Of Tlr4 In Macrophages Renderedmentioning

confidence: 99%

See 1 more Smart Citation

CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

Rajesh

Perkins

Ireland

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

125

104

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“…To establish stably transfected cells, TLR4/pEFBOS constructs were transfected into HEK293 cells with pBabePuro selection vectors using Lipofectamine 2000. After puromycin selection, stably transfected clones were screened by flow cytometry using UT49 or another TLR4 mAb, as described previously (26). Ba/F3 reporter cells carrying NF-kB-responsive luciferase gene (5) were transfected with a TLR4/pEFBOS construct with a MD-2/pCAGGS1 construct by the electroporation with GenePulser (Bio-Rad, Hercules, CA).…”

Section: Transfection Studymentioning

confidence: 99%

“…In this study, we dissected B cell activation using agonistic mAbs against TLR4 (26) and RP105 (16), in addition to LPS. Use of these agonistic mAbs provides a distinct advantage, because cross talk with TLR family members other than TLR4 and RP105 because of contaminated pathogen-derived stimulants is negligible.…”

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confidence: 99%

Reduced Surface Expression of TLR4 by a V254I Point Mutation Accounts for the Low Lipopolysaccharide Responder Phenotype of BALB/c B Cells

Tsukamoto

Fukudome²,

Takao³

et al. 2013

The Journal of Immunology

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LPS is recognized by TLR4 and radioprotective 105 kDa in B cells. Susceptibility to LPS in murine B cells is most closely linked to the locus containing the TLR4 gene. However, the molecular mechanism underlying genetic control of LPS sensitivity by this locus has not been fully elucidated. In this study, we revealed that C57BL/6 (B6) B cells respond to mAb-induced, TLR4-specific signals stronger than BALB/c (BALB) B cells, as assessed by proliferation and upregulation of CD69 and CD86. In contrast, BALB B cells were not hyporesponsive to agonistic anti–radioprotective 105 kDa mAb or the TLR9 agonist CpG. Although the level of TLR4 mRNA in BALB B cells was comparable with that in B6 B cells, surface TLR4 expression in BALB B cells was lower than that in B6 B cells. This lower surface expression of BALB TLR4 was also observed when HEK293 and Ba/F3 cells were transfected with a BALB TLR4 expression construct. We identified a V254I mutation as the responsible single nucleotide polymorphism for lower surface expression of BALB TLR4. Furthermore, cotransfection of myeloid differentiation factor-2 increased BALB TLR4 expression, although it was still lower than B6 TLR4 expression. In concordance with reduced expression, Ba/F3 cells transfected with BALB TLR4 and myeloid differentiation factor-2 were hyporesponsive compared with those with B6 TLR4, as assessed by LPS-induced NF-κB activation. In conclusion, we revealed that LPS sensitivity is genetically controlled by the level of surface TLR4 expression on B cells. A V254I mutation accounts for the LPS hyporesponsive phenotype of BALB B cells.

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Impaired antigen‐specific lymphocyte priming in mice after Toll‐like receptor 4 activation via induction of monocytic myeloid‐derived suppressor cells

et al. 2019

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In sepsis, the pathology involves a shift from a proinflammatory state toward an immunosuppressive phase. We previously showed that an agonistic anti‐TLR4 antibody induced long‐term endotoxin tolerance and suppressed antigen‐specific secondary IgG production when primed prior to immunization with antigen. These findings led us to speculate that TLR4‐induced innate tolerance due to primary infection causes an immunosuppressive pathology in sepsis. Therefore, the mechanism underlying impaired antigen‐specific humoral immunity by the TLR4 antibody was investigated. We showed, in a mouse model, that primary antigen‐specific IgG responses were impaired in TLR4 antibody‐induced tolerized mice, which was the result of reduced numbers of antigen‐specific GC B cells and plasma cells. Ovalbumin‐specific CD4 and CD8 T‐cell responses were impaired in TLR4 antibody‐injected OT‐I and ‐II transgenic mice ex vivo. Adoptive transfer studies demonstrated suppression of OVA‐specific CD4 and CD8 T‐cell responses by the TLR4 antibody in vivo. The TLR4 antibody induced Gr1+CD11b+ myeloid‐derived suppressor cell (MDSC) expansion with suppression of T‐cell activation. Monocytic MDSCs were more suppressive and exhibited higher expression of PD‐L1 and inducible nitric oxidase compared with granulocytic MDSCs. In conclusion, immune tolerance conferred by TLR4 activation induces the expansion of monocytic MDSCs, which impairs antigen‐specific T‐cell priming and IgG production.

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Preparation and Characterization of Agonistic Monoclonal Antibodies Against Toll-Like Receptor 4-MD-2 Complex

Cited by 17 publications

References 26 publications

CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

Reduced Surface Expression of TLR4 by a V254I Point Mutation Accounts for the Low Lipopolysaccharide Responder Phenotype of BALB/c B Cells

Impaired antigen‐specific lymphocyte priming in mice after Toll‐like receptor 4 activation via induction of monocytic myeloid‐derived suppressor cells

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