Antibodies of the immunoglobulin G1 course are induced in mice by

Antibodies of the immunoglobulin G1 course are induced in mice by T-cell-dependent antigens however, not by lipopolysaccharide (LPS). activating the promoter. Consequently, germ range 1 transcription depends upon the structure of LY2886721 NF-B/Rel protein. After activation by disease or immunization, naive relaxing B cells expressing IgM and IgD change to manifestation of IgG, IgE, and IgA isotypes. Isotype, or course, switching can be mediated with a DNA recombination event known as course change recombination. Recombination happens between two change areas, one located 5 towards the C gene as well as the additional located 5 to 1 from the downstream heavy chain constant region (CH) genes. Class switching does not alter the antigen specificity of the antibody isotype but does alter its effector function, e.g., the ability to bind to complement, to transcytose across epithelial cells, or to mediate an allergic reaction (41, 44). Cytokines, B-cell mitogens, and the nature of antigen determine the choice of isotype. For example, the IgG1 and IgG3/IgG2b antibody classes are preferentially induced in mice by T-cell-dependent and -independent antigens, e.g., proteins and bacterial LPS, respectively (31, 40). An initiating event in class switching is the induction of transcription of the unrearranged, or GL, CH gene to which the cell will switch. Transcription from the GL CH gene initiates at exon I, upstream of the switch region of each CH gene (44). Subsequent RNA splicing to produce mature GL transcripts, also called switch transcripts, appears to be required for switch recombination (26). The effect of targeted disruption of the promoter and exon I of GL CH gene transcripts provides solid evidence for the requirement for GL transcripts in class switching (4, 20, 54). Since GL transcripts direct switch recombination, an understanding of the mechanisms of regulation of GL transcripts is necessary for understanding the regulation of class switching. Expression of GL transcripts is regulated at LY2886721 the transcriptional level by cytokines, such as IL-4, gamma interferon, and transforming growth factor , and by B-cell activators, such as LPS, CD40L, and stimuli that induce signaling through surface Ig (19, 27, 37, 44, 45, 48, 53). For example, IL-4 and CD40L induce GL 1 transcripts, whereas LPS, LY2886721 in the absence of IL-4, induces GL 2b and 3 transcripts (27, 37, 48). We previously reported that CD40L, but not LPS, induces the activity of the GL 1 promoter in a reporter plasmid (25). This induction by CD40L is mediated by activating NF-B/Rel proteins to bind to three tandem B sites located in the CD40RR of the GL 1 promoter, which is located just Rabbit Polyclonal to TISB (phospho-Ser92). downstream of an IL-4-responsive Stat6 binding site (3). Overexpression of NF-B/p50 together with RelA or RelB is sufficient to transactivate the GL 1 promoter or the CD40RR inserted upstream of a minimal c-promoter, whereas overexpression of NF-B/p50 together with c-Rel fails to induce even though this heterodimer binds to the GL 1 promoter. These observations suggest that NF-B/Rel proteins are important for initiating class switching to IgG1 in response to T-dependent antigens. Although LPS is an activator of NF-B, LPS is a poor inducer of GL 1 transcripts. In this study, we have addressed the reason for the differential effects of LPS and CD40L on induction of GL 1 promoter and transcripts. We have tested the hypothesis that GL 1 transcription depends on the composition of the induced NF-B/Rel dimers. MATERIALS AND METHODS Abbreviations. Abbreviations used in this paper are as follows: Ig, immunoglobulin; GL, germ line; CD40L, CD40 ligand; CD40RR, CD40 responsive region; LPS, lipopolysaccharide; IL, interleukin; PDTC, pyrrolidine dithiocarbamate; TLCK, and v-serotype O55:B5), NF-B inhibitors PDTC and TLCK, and an NF-B activator (okadaic acid) were purchased from Sigma. TLCK and okadaic acid were dissolved in DMSO at 75 mM and at 125 g/ml, respectively, and were diluted 1,000-fold when added to cells. LPS and PDTC were dissolved in RPMI medium. RNA isolation. Total RNA was prepared from 1B4.B6 and splenic B cells by the hot phenol method (33). Semiquantitative RT-PCR. (i) RT. RT was performed with the following specific primers: 5-CTGAGCTGCTCAGAGTGTA-3 (positions 300 to 282, GenBank no. V00793) (18) for GL 1 transcripts and 5-TCACAAACATGGGGGCATC-3 (positions 437 to 419, GenBank no. “type”:”entrez-nucleotide”,”attrs”:”text”:”M32599″,”term_id”:”193423″,”term_text”:”M32599″M32599) (35) for GAPDH transcripts. Both of these specific primers had been blended with 4 g of total RNA from splenic B cells or 30 g of total RNA from 1B4.B6 cells. The blend was warmed at 65C for 10 min and cooled off to room temperatures..