Hydrogen peroxide modulates immunoglobulin expression by targeting the 3′Ighregulatory region through an NFκB-dependent mechanism

Abstract

Reactive oxygen species such as hydrogen peroxide (H2O2) appear to play a role in signal transduction in immune cells and have been shown to be synthesized upon antigen-mediated activation and to facilitate cellular activation in B and T cells. However an effect of H2O2 on B-cell function (i.e. immunoglobulin (Ig) expression) has been less well-characterized. The effects of H2O2 exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in Ig heavy chain (Igh) gene expression. Igh transcription in B lymphocytes is an essential step in antibody production and is governed through a complex interaction of several regulatory elements, including the 3’Igh regulatory region (3’IghRR). Utilizing an in vitro mouse B-cell line model, this study demonstrates that exposure to low μM concentrations of H2O2 can enhance 3’IghRR-regulated transcriptional activity and Igh gene expression, while either higher concentrations of H2O2 or the expression of a degradation resistant inhibitory κB (IκBα super-repressor) can abrogate this effect. Furthermore, suppressive H2O2 concentrations increased protein levels of the p50 NFκB subunit, IκBα, and an IκBα immunoreactive band which was previously characterized as an IκBα cleavage product exhibiting stronger inhibitory function than native IκBα. Taken together, these observations suggest that exposure of B lymphocytes to H2O2 can alter Igh transcriptional activity and Ig expression in a complex biphasic manner which appears to be mediated by NFκB and altered 3’IghRR activity. These results may have significant implications to disease states previously associated with the 3’IghRR.

The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3′ Immunoglobulin Heavy Chain Regulatory Region

“…To initiate studies that directly evaluate the role of NF-jB/Rel proteins in the effects of LPS and TCDD on 3 0 IghRR activity, we utilized another variant of the CH12.LX cell line (ie, CH12.IjBaAA). The CH12.IjBaAA cell line stably expresses an IPTG-inducible IjBa superrepressor protein (IjBaAA), which is resistant to negative feedback regulation by NF-jB/Rel proteins (Hsing and Bishop, 1999;Romer and Sulentic, 2011). We verified the inducibility of IjBaAA expression, by treating the CH12.IjBaAA cells with varying concentrations of IPTG overnight (approximately 17 h) or with 100 lM IPTG from 0 to 5 h followed by whole-cell protein isolation and Western blot analysis.…”

“…The RNA concentration was determined using a NanoDrop (ThermoScientific, Wilmington, Delaware) and 200 ng total RNA was reverse transcribed to cDNA using the Taqman Reverse Transcription Reagents kit (Applied Biosystems, Foster City, California). The expression of b-actin (endogenous control to normalize cDNA concentrations) and cytochrome P4501a1 (Cyp1a1) genes was quantified by real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems) as previously described (Fernando et al, 2012;Romer and Sulentic, 2011). Primers for Cyp1a1 and b-actin span an intron and were as follows: Cyp1a1 Forward Primer-AAGTGCAGATGCGGTCTTCT, Cyp1a1 Reverse Primer-AAAGTAGGAGGCAGGCACAA, b-actin Forward Primer-GCTACAGCTTCACCACCACA, and b-actin Reverse Primer-TCTCCAGGGAGGAAGAGGAT.…”

“…The samples were incubated for a minimum of 5 h, cooled to 45 C, and treated with 1 ml of 10 mg/ml proteinase K for 2 h. The DNA from the samples was extracted with phenol/chloroform extraction, and the presence of DNA was validated with a NanoDrop Spectrophotometer. SYBRGreen real-time PCR was utilized to analyze the DNA samples (2 ml) as previously described (Romer and Sulentic, 2011) with the exception of using the CFX96 Touch Real-Time PCR System (Bio-Rad). PCR primers were as follows: hs4 Forward Primer-CACA CCCCACCTGTAGCAC, hs4 Reverse Primer-TGAGGAGGTTGAC ATGATGG, hs1.2 Forward Primer-CTGATATCTGAGCCCCCAAC, hs1.2 Reverse Primer-GTGGTCTGGGTAATGGATGG, b-actin Forward Primer-GCTACAGCTTCACCACCACA, and b-actin Reverse Primer-TCTCCAGGGAGGAGGAGGAT.…”

The aryl hydrocarbon receptor regulates an essential transcriptional element in the immunoglobulin heavy chain gene

“…RNA was isolated as before [24]. Briefly, samples were thawed at room temperature, mixed with 0.1 volume of 1-bromo-3-chloropropane, and the aqueous phase separated using Phase Lock Gel Heavy Tubes (5 PRIME, Gaithersburg, MD).…”

2,3,7,8-Tetrachlorodibenzo-p-Dioxin Induces Transcriptional Activity of the Human Polymorphic hs1,2 Enhancer of the 3′Igh Regulatory Region

“…The expression of β- actin (endogenous control to normalize cDNA concentrations) and the endogenous heavy- and light-chain (i.e. α constant region, C α and κ constant region, C κ, respectively) genes were quantified by real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems) as previously described (42). Primers for Cα, Cκ, and β- actin span an intron and are listed in Table 1.…”