Primary B cells essentially lack constitutive NF-κB activity

Yedidia, Yifat; Ben‐Neriah, Yinon

doi:10.1002/(sici)1521-4141(199801)28:01<30::aid-immu30>3.0.co;2-h

Cited by 6 publications

(7 citation statements)

References 45 publications

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“…This would be in agreement with previous studies with 70Z/ 3 cells, where TGF-L treatment of LPS-stimulated cells blocked both U gene and Oct-2 expression but not NF-UB activation, demonstrating that NF-UB by itself is necessary but not su¤cient to induce U gene expression [24]. Our failure to detect NF-UB in mature 70Z/3 cells is consistent with the ¢nding that primary splenic mouse B cells lack constitutive NF-UB binding activity [25], suggesting that the role of NF-UB in B cells could be important for developmental cellular processes but not for the maintenance of the di¡erentiated phenotype.…”

Section: Discussionsupporting

confidence: 93%

Possible stage‐specific function of NF‐κB during pre‐B cell differentiation

1998

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Lipopolysaccharide (LPS)-induced differentiation of the murine pre-B cell line 70Z/3 is a model for pre-B to B cell differentiation and has been used to show that the transcription factor NF-U UB is essential to induce the expression of the IgU U gene. We have investigated the mechanism involved in late stages of the process when all cells have reached a more mature B phenotype, i.e. beyond 48 up to 96 h of LPS treatment. NF-U UB binding activity was induced at early times by LPS treatment, but its DNA binding activity disappeared after 84 h of LPS treatment. Accumulation of IU UBK K protein in the nucleus correlated with the disappearance of NF-U UB activity at 72, 84 and 96 h, and treatment of nuclear extracts of 72^96 h LPStreated cells with Na-deoxycholate restored NF-U UB binding activity. The data indicate that NF-U UB, while important to initiate the process of IgU U gene transcription in 70Z/3 pre-B cells, is no longer required for its maintenance in differentiated 70Z/3 cells.z 1998 Federation of European Biochemical Societies.

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Section: Discussionsupporting

confidence: 93%

Possible stage‐specific function of NF‐κB during pre‐B cell differentiation

1998

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“…Nuclear extraction was conducted as previously described. 29 Briefly, LPS-stimulated B cells, irradiated or nonirradiated, were washed and resuspended in 200-400 ll hypotonic buffer containing 10 mm Hepes (pH 7AE9), 0AE1 mm ethylenediaminetetraacetic acid (EDTA), 0AE1 mm egtazic acid (EGTA), 10 mm KCl, 1 mm dithiothreitol (DTT), 0AE75 mm spermidine, 0AE15 mm spermine, 20 mm p-nitrophenyl phosphate, 20 mm b-glycerophosphate, 1 mm Na 3 VO 4 , and protease inhibitors (1 mm phenylmethylsulphonyl fluoride, 5 lg ⁄ml leupeptin, and 5 lg ⁄ml peptstatin). After 15 min on ice, 0AE6% NP40 was added and the lysates were further incubated for an additional 5 min and were centrifuged at 13 000 g in a microcentrifuge at 4°.…”

Section: Electrophoretic Mobility Shift Assay (Emsa)mentioning

confidence: 99%

“…For determination of NF-jB activity in nuclear extract, an EMSA was performed as previously described. 29 A synthetic oligonucleotide probe containing nucleotides 2969-2945 of the human CD80 enhancer (5¢-GGGAAAGGGGTTTTCCCAGCAGTCA-3¢), which includes the NF-jB binding site. 28 The probe was annealed with a synthetic nucleotide 5¢-TGACTGCTGGGAAAA CCCCTTT-3¢ to form double-stranded DNA.…”

Section: Electrophoretic Mobility Shift Assay (Emsa)mentioning

confidence: 99%

Irradiation up‐regulates CD80 expression through two different mechanisms in spleen B cells, B lymphoma cells, and dendritic cells

Torihata

Ishikawa²,

Okada³

et al. 2004

Immunology

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SUMMARYWe have previously demonstrated irradiation-induced up-regulation of CD80 expression in A20-HL B lymphoma cells by inducing expression of tumour necrosis factor-a (TNF-a) and CD154. In the present study, we investigated whether irradiation also up-regulates CD80 expression in mouse spleen B cells. Because freshly prepared spleen B cells are highly sensitive to irradiation, we employed spleen B cells stimulated with lipopolysaccharide (LPS-B cells). X-irradiation (8 Gy) followed by incubation (9-12 hr) highly and selectively up-regulated CD80 expression in LPS-B cells, whereas the same treatment slightly increased expression of CD54 and did not affect expression of CD86, major histocompatibility complex class II, CD11a or surface immunoglobulin M. The irradiation-induced up-regulation of CD80 expression resulted in enhanced APC function of LPS-B cells. Up-regulation of CD80 expression on LPS-B cells was accompanied by an increase in CD80 mRNA accumulation and nuclear factor (NF)-jB activation. Activation of NF-jB was shown to be critical for up-regulation of CD80 expression as pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-jB, severely decreased the observed up-regulation. X-irradiation of LPS-B cells induced expression of TNF-a but not CD154. However, anti-TNF-a monoclonal antibody (mAb) with anti-CD154 mAb did not inhibit X-irradiation-induced up-regulation of CD80 expression in LPS-B cells, whereas these mAbs almost completely inhibited this up-regulation in A20-HL cells and bone marrow-derived dendritic cells (DCs). In contrast, a thiol antioxidant, N-acetyl-L-cysteine, completely blocked X-irradiation-induced up-regulation of CD80 expression in LPS-B cells, but not in A20-HL cells or in DCs. Based on these findings, we concluded that X-irradiation up-regulates CD80 expression not only in A20-HL cells and DCs but also in LPS-B cells, and that this up-regulation in LPS-B cells via NF-jB activation is dependent on the generation of reactive oxygen species, while that in A20-HL cells and DCs is not.

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“…To minimize potential in vitro activation of NF-B during cell preparation (17), all isolation procedures were performed at 4°C or on ice. Spleens isolated from 1-to 2-mo-old female C57BL/6 mice housed at the pathogenfree University of Wisconsin Comprehensive Cancer Center Animal Facility were kept on ice, single cells were manually released, and RBCs were hypotonically lysed with ice-cold water followed by immediate isotonic adjustment with ice-cold 10ϫ PBS.…”

Section: Preparation and Fluorescent Activated Cell Sorting Of Primarmentioning

confidence: 99%

“…To determine whether a novel proteasome-independent pathway mediates NF-B activity in vivo, we isolated primary B lymphocytes from murine spleen while minimizing the potential in vitro activation (17) by maintaining the isolation procedures at 4°C. In agreement with prior reports (5, 6), we found that the majority of constitutive NF-B DNA-binding activity observed in primary splenocytes is derived from the B cell compartment (data not shown).…”

Section: Maintenance Of Constitutive Nf-b Activity In Primary Splenocmentioning

confidence: 99%

A Switch in Distinct IκBα Degradation Mechanisms Mediates Constitutive NF-κB Activation in Mature B Cells

Fields

Seufzer

Oltz

et al. 2000

The Journal of Immunology

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Inducible activation of cytoplasmic NF-κB/Rel transcription factors occurs via proteasome-dependent degradation of an associated inhibitor, termed IκBα. Mature B lymphocytes constitutively express nuclear NF-κB, which is important for their long-term survival. The intrinsic mechanisms by which B cells constitutively activate NF-κB are unknown. In this paper we demonstrate that maintenance of NF-κB activity in primary B cells is mediated by a novel calcium-dependent, but proteasome-independent, mechanism. Moreover, we show that differentiation of conditionally transformed pre-B cells is accompanied by a switch from proteasome-dependent to proteasome-independent degradation of IκBα. Our findings indicate that IκBα degradation mechanisms are dynamic during B cell development, and ultimately establish constitutive NF-κB activity in mature B lymphocytes.

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Primary B cells essentially lack constitutive NF-κB activity

Cited by 6 publications

References 45 publications

Possible stage‐specific function of NF‐κB during pre‐B cell differentiation

Possible stage‐specific function of NF‐κB during pre‐B cell differentiation

Irradiation up‐regulates CD80 expression through two different mechanisms in spleen B cells, B lymphoma cells, and dendritic cells

A Switch in Distinct IκBα Degradation Mechanisms Mediates Constitutive NF-κB Activation in Mature B Cells

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