Induction of Long-Term Lipopolysaccharide Tolerance by an Agonistic Monoclonal Antibody to the Toll-Like Receptor 4/MD-2 Complex

Abstract

We have established an agonistic monoclonal antibody, UT12, that induces stimulatory signals comparable to those induced by lipopolysaccharide (LPS) through Toll-like receptor 4 and MD-2. UT12 activated nuclear factor B and induced the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-␣) and interleukin-6 (IL-6) in peritoneal exudative cells. In addition, mice injected with UT12 rapidly fell into endotoxin shock concomitant with the augmentation of serum TNF-␣ and IL-6 levels, followed by death within 12 h. On the other hand, when the mice were pretreated with a sublethal dose of UT12, the mice survived the subsequent lethal LPS challenges, with significant suppression of serum TNF-␣ and IL-6, indicating that UT12 induced tolerance against LPS. This effect of UT12 was maintained for at least 9 days. In contrast, the tolerance induced by LPS continued for less than 3 days. These results illuminate a novel potential therapeutic strategy for endotoxin shock by the use of monoclonal antibodies against the Toll-like receptor 4/MD-2 complex.Lipopolysaccharide (LPS) is a glycolipid component of the gram-negative bacterial cell wall and induces various host responses, including the production of proinflammatory cytokines. When they are appropriately produced, these cytokines, such as tumor necrosis factor alpha (TNF-␣) and interleukin-6 (IL-6), activate host immunity to fight off bacteria. The excessive proinflammatory cytokines produced in response to large amounts of LPS, however, can provoke extreme systemic inflammation and often cause lethal endotoxin shock.Animals pretreated with a sublethal dose of LPS become tolerant to subsequent challenges with a lethal dose of LPS and display reduced mortality. This phenomenon is called LPS tolerance and is defined as the reduced capacity of the host or cultured macrophage/monocyte to respond to LPS following initial stimulation (6,26). It has also been reported that bacterial or fungal removal is improved during the tolerant state, despite attenuated cytokine production (14,20). Therefore, LPS tolerance is regarded as a reasonable response that simultaneously manages both the clearance of pathogens and host protection from excess inflammation.Here we report on the induction of long-term LPS tolerance realized by an agonistic monoclonal antibody (MAb) against the Toll-like receptor 4 (TLR4)/MD-2 complex. Mice pretreated with this MAb showed significant survival advantages compared with the survival of LPS-pretreated mice. MATERIALS AND METHODSMice. C3H/HeN, C3H/HeJ, ddY, and SCID mice were from Japan SLC (Hamamatsu, Shizuoka, Japan). C57BL/6 mice were from Charles River Japan (Yokohama, Kanagawa, Japan). A TLR4-knockout mouse strain with the C57BL/6 background (12) was a kind gift from S. Akira (Osaka University, Osaka, Japan). All animals were maintained in the Center for Laboratory Animals at Saga Medical School and were treated in accordance with the regulations of the Scientists Center for Animal Welfare.Cell culture. All the cells were cult...

Accelerated Prion Disease Pathogenesis in Toll-Like Receptor 4 Signaling-Mutant Mice

“…While administration of TLR4 ligands directly to the CNS may effectively induce clearance of amyloid (19), in practice this route would be complicated to exploit in humans. The alternative, systemic treatment with these agents by peripheral injection, could lead to lethal side effects induced by high levels of evoked proinflammatory mediators, such as TNF-␣ and IL-1␤ (17,44). The negative consequences of TNF-␣ and IL-1␤ generation, which result from TLR4 activation, might be counteracted without dampening prion infection-protective phagocyte activation by coadministering specific antagonists to neutralize these cytokines (4,27,39).…”

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

“…It has been shown that UT12 induces endotoxic shock-like symptoms in mice including augmentation of TNF-α and IL-6. Furthermore, UT12 induced long-term tolerance and protection against LPS-induced lethal shock in mice (14). However, the ability of UT12 to induce translocation of TLR4/MD2 into endosomes, as well as its potential for mediating TRIF-dependent signaling, has not been reported.…”

“…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).…”

Toll-Like Receptor 4 Agonistic Antibody Promotes Innate Immunity against Severe Pneumonia Induced by Coinfection with Influenza Virus and Streptococcus pneumoniae

“…UT12 acts as an agonist of the TLR4/MD-2 complex and induces a stimulatory signal similar to the original ligand LPS (15). UT12 can induce the production of NF-B and inflammatory cytokines involved in the innate immune system from peritoneal exudate cells in vitro (15). Previous studies have demonstrated that prophylactic treatment with TLR ligands enhances host immunity against influenza virus infection or pneumococcal infection alone (16,17).…”

“…UT12 acts as an agonist of the TLR4/MD-2 complex and induces a stimulatory signal similar to the original ligand LPS (15). UT12 can induce the production of NF-B and inflammatory cytokines involved in the innate immune system from peritoneal exudate cells in vitro (15).…”