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purpose. IκBζ−/− mice have been reported to be affected by allergic dermatitis. This study was conducted to analyze the pathophysiological role of IκBζ and to address the functional relevance of Th2-mediated immune responses in the development of ocular surface inflammation and dermatitis by IκBζ−/− mice. methods. BALB/c background IκBζ−/− mice were established without individual differences; IκBζ/Stat6 double-knockout (WKO) mice unable to produce Th2 cytokine were created; and microscopic-, histologic-, and immunochemical studies were performed. In IκBζ−/− mice the serum IgE levels were examined by ELISA, and quantitative PCR was used to study the gene expression of IFN-γ, IL4, IL10, TNFα, IL6, IL17α, and CCL11 in eyelid tissue. results. IκBζ−/− mice exhibited a severe inflammatory phenotype on the ocular surface and perioral skin. The inflammatory infiltrates in the perioral skin consisted primarily of CD4+ and CD8+ cells; CD4+ and CD45R/B220+ cells were mainly detected in the conjunctiva. In eyelid and perioral skin tissue, the expression of IL-17α and of Th1 and Th2 cytokines, but not of CCL11, was augmented. IκBζ−/− and IκBζ+/− mice did not differ significantly in their serum total IgE levels before, 0 to 4 weeks, and 5 to 9 weeks after disease onset. IκBζ/Stat6 WKO mice showed the same or slightly more severe inflammation than did IκBζ−/− mice. conclusions. IgE and Stat6 are not responsible for the immune pathologic response leading to the development of ocular surface and perioral skin inflammation in IκBζ−/− mice. IκBζ−/− mice may be a suitable model for Stevens-Johnson syndrome, but not for atopic dermatitis.
No abstract is provided for this article.
of Microbiology and Immunology University of California Berkeley, California 94720 Summary DNA-nuclear protein interactions were studied with synthetic recombination signal sequences (RSSs) for immunoglobulin V-J joining. With a gel retardation as- say, a DNA-binding protein that specifically interacts with RSSs was detected in nuclear extracts from a pre- B cell line, 3889. This protein was found in all the recombination-competent pre-B cell lines tested in this study, but not in myeloma, mature T cell, mono- cyte, or fibroblast cell lines. DNA footprint analysis with dimethyl sulfate demonstrated that the 7-mer re- gion of the RSS was strongly protected when com- plexed with the binding protein. Furthermore, a single base substitution in the Fmer region totally abolished the binding. The molecular mechanism of V-J joining is discussed in the context of the RSS-binding protein. Introduction Antigen receptor genes are generated by site-specific DNA recombination both in T cells and 6 cells during the differentiation of lymphocytes (reviewed by Kronenberg et al., 1986; Tonegawa, 1983). On the germ-line chromo- some, variable region genes for the antigen receptor are split into variable (V), diversity (D), and joining (J) seg- ments. These gene segments are brought together by a site-specific DNA recombination process known as V-(D)- J joining. This DNA rearrangement plays a key role not only in the activation of the antigen receptor genes, but also in the diversification of the gene sequences. To study the molecular mechanism of V-(D)-J joining, three different approaches have been taken. One ap- proach is the structural study of the recombination region by DNA cloning and sequencing. From these analyses it has been revealed that both immunoglobulin (lg) and T cell receptor (TCR) genes contain two highly conserved sequences adjacent to the recombination sites; they are a palindromic 7-mer, CACTGTG, and a T-rich 9-mer, GGTTTTTGT, separated by a spacer of constant length (Sakano et al., 1979, 1980, 1981; Max et al., 1979; Early et al., 1980; Davis, 1985). The second approach is to in- troduce artificial recombination substrates with appropri- ate selection markers into recombination-competent pre-B cells (Lewis et al., 1984, 1985; Blackwell and Alt, 1984; Yancopoulos et al., 1986; Hesse et al., 1987). Alt and Balti- more and their colleagues have demonstrated that V-(D)-J joining can take place on exogenous lg or TCR genes in- troduced into the host chromosomal DNA with a retroviral vector or by DNA transfection. The third approach is to characterize the enzymatic machinery responsible for V-(D)-J joining. We assume that at least three activities are involved in the recombinase complex for V-(D)-J joining (Hope et al., 1986): a DNA-binding activity, an endonu- cleolytic activity, and a ligase activity. The DNA-binding activity would recognize the recombination sites and bring the two substrates together into the proper orientation; the endonucleolytic activity would cleave the germ-line seg- ments at the recombination sites; and finally, the ligase activity would join the two cleaved DNA substrates cova- lently to complete the joining reaction. Endonucleases presumably involved in the DNA-cutting activity of recombinase have been previously described (Desiderio and Baltimore, 1984; Kataoka et al., Hope 1986). However, little is known about other activities, and as yet the identification of a whole recombinase com- plex has not been reported. In the present study we have attempted to identify a DNA-binding protein(s) that interacts specifically with recombination signal sequences (RSSs). As a source of the protein, we have used nuclear extracts from an immature pre-B cell line, 3889, kindly provided by Dr. F. W. Alt. In this cell line lg gene rearrangement actively takes place during in vitro culture (Alt et al., 1981; Black- well and Alt, 1984). We synthesized DNA substrates con- taining the Fmer and 9-mer separated by either a 12 bp or a 23 bp spacer. Using the synthetic recombination sub- strates, we first verified that two sets of RSSs are sufficient to cause the V-J-type joining provided the 12/23 bp spacer rule (Sakano et al., 1980; Early et al., 1980) is satisfied. We then searched for a specific DNA-binding protein(s) in the pre-B cell nuclear extracts by using the synthesized RSS probes. With a gel retardation assay (Singh et al., 1986; Schneider et al., 1986), we have identified a nuclear pro- tein that can specifically interact, particularly in the Fmer region, with the RSS probe containing the 12 bp spacer. In this report we characterize this RSS-binding activity, which may be one of the DNA-binding components as- sociated with V-(D)-J joining. Results Synthetic Substrates Containing the Fmer and 9-mer Can Cause V-J-Type Recombination Two double-stranded oligonucleotides containing the Fmer and 9-mer were chemically synthesized (Figure 1A). One contained a 12 bp spacer separating the recombina- tion signals (12 bp-RSS), and the other contained a 23 bp spacer (23 bp-RSS). To study whether these two RSSs are sufficient substrates to mediate V-J-type recombina- tion, we constructed a plasmid, pKOdel-1. It contains a ne- omycin resistance gene (near) (Beck et al., 1982) flanked by two sets of RSSs, and a thymidine kinase gene (tk) (McKnight, 1980) further downstream (Figure 1B). Expres- sion of these genes is mediated by a promotor complex consisting of the SV40 and t/r gene promoters (McKnight, 1980; Southern and Berg, 1982).
No abstract is provided for this article.
Toll-like receptors (TLRs) play a critical role in the detection of invading pathogens and subsequent immune response against them. Individual TLRs recognize distinct microbial components. The TLR is a type 1 transmembrane receptor that is composed of an extracellular leucine-rich repeat (LRR) domain and cytoplasmic domain homologous to that of the IL-1R family. Therefore, TLR and IL-1R family use the same signaling molecules. Cytokine production in response to each TLR ligand is completely abolished in MyD88-deficient cells, indicating that MyD88 is an essential signaling molecule shared among IL-1R/Toll family. However, accumulating evidence indicates that differential utilization of adaptors including MyD88, TIRAP, and TRIF may activate overlapping as well as distinct signaling pathways, and finally give rise to distinct biological effects exerted by individual TLR family.
