IL-1 receptor-associated kinase modulates host responsiveness to endotoxin.

Endotoxin triggers many of the inflammatory, hemodynamic, and hematological derangements of Gram-negative septic shock. Recent genetic studies in mice have identified the Toll-like receptor 4 as the transmembrane endotoxin signal transducer. The IL-1 intracellular signaling pathway has been implicated in Toll-like receptor signal transduction. LPS-induced activation of the IL-1 receptor-associated kinase (IRAK), and the influence of IRAK on intracellular signaling and cellular responses to endotoxin has not been explored in relevant innate immune cells. We demonstrate that LPS activates IRAK in murine macrophages. IRAK-deficient macrophages, in contrast, are resistant to LPS. Deletion of IRAK disrupts several endotoxin-triggered signaling cascades. Furthermore, macrophages lacking IRAK exhibit impaired LPS-stimulated TNF-alpha production, and IRAK-deficient mice withstand the lethal effects of LPS. These findings, coupled with the critical role for IRAK in IL-1 and IL-18 signal transduction, demonstrate the importance of this kinase and the IL-1/Toll signaling cassette in sensing and responding to Gram-negative infection.

Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase.

Stimulation of the type 1 IL-1R (IL-1R1) and the IL-18R by their cognate ligands induces recruitment of the IL-1R-associated kinase (IRAK). Activation of IRAK leads in turn to nuclear translocation of NF-kappaB, which directs expression of innate and adaptive immune response genes. To study IRAK function in cytokine signaling, we generated cells and mice lacking the IRAK protein. IRAK-deficient fibroblasts show diminished activation of NF-kappaB when stimulated with IL-1. Immune effector cells without IRAK exhibit a defective IFN-gamma response to costimulation with IL-18. Furthermore, mice lacking the Irak gene demonstrate an attenuated response to injected IL-1. Deletion of Irak, however, does not affect the ability of mice to develop delayed-type hypersensitivity or clear infection with the intracellular parasite, Listeria monocytogenes. These results demonstrate that although IRAK participates in IL-1 and IL-18 signal transduction, residual cytokine responsiveness operates through an IRAK-independent pathway.

Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice.

The MHC class I-related receptor, FcRn, mediates the transfer of maternal gamma globulin (IgG) to young rodents, primarily via intestinal transcytosis, and this provides humoral immunity for the first few weeks after birth. In a previous study, the site of mouse IgG1 (mIgG1) with which FcRn interacts has been mapped using recombinant wild-type and mutated Fc-hinge fragments. The site encompasses residues at the CH2-CH3 domain interface of Fc (Ile253, His310, Gln311, His433 and Asn434) and the same amino acids are involved in regulating the pharmacokinetics of the Fc-hinge fragments. This suggests that in addition to its known function, FcRn might also play a role in IgG homeostasis. Consistent with this hypothesis, in this study, we demonstrate that FcRn alpha-chain mRNA is present not only in neonatal brush border but also in other tissues of adult animals (liver, lung, spleen and endothelial cells). In addition, analysis of the pharmacokinetics of mouse Ig/Fc-hinge fragments in genetically manipulated mice that are deficient in the expression of FcRn demonstrates that the beta-phase half-lives are abnormally short. These findings suggest that FcRn is involved in IgG homeostasis.

Evidence that the hinge region plays a role in maintaining serum levels of the murine IgG1 molecule.

The site of the murine IgG1 molecule that regulates catabolism has recently been shown to encompass amino acids that are located at the CH2-CH3 domain interface. The CH2 and CH3 domains are connected to each other by a relatively flexible "mini-hinge" region, and flexibility in this region could clearly affect the orientation of the domains with respect to each other. The internal movement of the CH2 domain depends on the absence/presence of the hinge disulphide. The increased mobility of the CH2 domain relative to the CH3 domain in a hinge less IgG or Fc fragment may result in a conformational change at the CH2-CH3 domain interface and alter the accessibility of the residues that are involved in catabolism control. To investigate this possibility, four Fc fragments which differ in the presence/absence of hinge disulphides and hinge sequences have been analysed in both in vivo pharmacokinetic studies and in vitro by limited proteolysis with pepsin. The data show that the presence of hinge disulphide(s) in the Fc fragment results in a longer intravascular half life but a higher susceptibility to pepsin attack. This, taken together with the knowledge that pepsin cleaves close to the CH2-CH3 domain interface, suggests that the longer half life of disulphide linked Fc fragments relative to unlinked fragments may be due to conformational differences in this region of the IgG molecule, and these conformational changes may affect the accessibility of the catabolic site for binding to putative protective Fc receptors.

Localization of the site of the murine IgG1 molecule that is involved in binding to the murine intestinal Fc receptor.

Site-directed mutagenesis of a recombinant Fc hinge fragment has recently been used to localize the site of the murine IgG1 molecule that is involved in the control of catabolism (the "catabolic site"). In the current study, the effects of these CH2 and CH3 domain mutations (Ile 253 to Ala 253, His 310 to Ala 310, Gln 311 to Asn 311, His 433 to Ala 433 and Asn 434 to Gln 434) on intestinal transfer of Fc hinge fragments in neonatal mice have been analyzed. Studies using direct transfer and competition assays demonstrate that the mutations affect the transmission from intestinal lumen into serum in a way that correlates closely with the effects of the mutations on pharmacokinetics. Binding studies of several of the Fc hinge fragments to isolated neonatal brush borders have been used to confirm the in vivo transmission data. These analyses have resulted in the localization of the binding site for the intestinal transfer receptor, FcRn, to specific residues of the murine Fc hinge fragment. These residues are located at the CH2-CH3 domain interface and overlap with both the catabolic site and staphylococcal protein A (SpA) binding site. The pH dependence of IgG1 or Fc fragment binding to FcRn is consistent with the localization of the FcRn interaction site to a region of the Fc that encompasses two histidine residues (His 310 and His 433). To assess whether one or two FcRn binding sites per Fc hinge are required for intestinal transfer, a hybrid Fc hinge fragment comprising a heterodimer of one Fc hinge with the wild-type IgG1 sequence and a mutant Fc hinge with a defective catabolic site (mutated at His 310, Gln 311, His 433 and Asn 434) has been analyzed in direct and competition transmission assays. The studies demonstrate that the Fc hybrid is transferred with significantly reduced efficiency compared to the wild type Fc hinge homodimer and indicate that the binding to FcRn, and possibly subsequent transfer, is enhanced by the presence of two FcRn binding sites per Fc hinge fragment.

Catabolism of the murine IgG1 molecule: evidence that both CH2-CH3 domain interfaces are required for persistence of IgG1 in the circulation of mice.

Site-directed mutagenesis of a recombinant Fc-hinge fragment has previously been used to identify a region of the murine IgG1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains. In the current study the nature of this 'catabolic site' has been further analysed using recombinant techniques. Fc-hinge, CH2-hinge, CH2 and CH3 fragments have been expressed in Escherichia coli, purified and analysed in pharmacokinetic studies in mice. The CH2-hinge has been analysed as both a monomer and dimer, and the dimer has a longer beta phase half-life (61.6 h) than the monomer (29.1 h). This suggests that two catabolic sites per Fc fragment are required for serum persistence. The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc-hinge fragment comprising a heterodimer of one Fc-hinge with the wild type (WT) IgG1 sequence and a mutant Fc-hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434). This hybrid is cleared with a beta phase half-life of 37.9 h and this is significantly shorter than that of the WT Fc-hinge fragment (82.9 h). In contrast to the CH2-hinge dimer, the CH3 domain is cleared rapidly (beta phase half-life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment. The data extend our earlier observations concerning a region of the murine IgG1 molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy.

Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis.

Site-directed mutagenesis has been used to change amino acid residues of a recombinant Fc-hinge fragment derived from the murine immunoglobulin (Ig)G1 molecule, and the effects of these mutations on the pharmacokinetics of the Fc-hinge fragment have been determined. Specifically, Ile-253, His-310 and Gln-311 of the CH2 domain and His-433 and Asn-434 of the CH3 domain have been changed. In the three dimensional structure of an antibody, these amino acids are in close proximity to each other at the CH2-CH3 domain interface. The mutated Fc-hinge fragments have been purified from recombinant Escherichia coli cells and their pharmacokinetic parameters determined in mice and compared with those of the wild-type Fc-hinge fragment. The results show that the site of the IgG1 molecule that controls the catabolic rate (the 'catabolic site') is located at the CH2-CH3 domain interface and overlaps with the Staphylococcal protein A binding site.