Interleukin-6 production by murine B cells and B cell lines

doi:10.1016/0008-8749(91)90041-9

Cellular Immunology

Volume 132, Issue 2, February 1991, Pages 442-450

https://doi.org/10.1016/0008-8749(91)90041-9 Get rights and content

Abstract

We have analyzed interleukin (IL)-6 gene transcription and IL-6 secretion by murine B cells in vitro. Mitogenic doses of lipopolysaccharide (LPS) or LPS in combination with F(ab′)₂ goat anti-mouse IgM antibodies (GAMμ), but not GAMμ alone, induced B cells to synthesize and release IL-6. In time course experiments, the accumulation of IL-6 mRNA was first detectable at 24–36 hr of culture and the levels were maintained through 60 hr; these kinetics correlated well with increases in supernatant IL-6 levels and were coincident with vigorous cell cycle activity. We also analyzed constitutive and LPS-induced IL-6 gene expression by the murine B cell lines: 70Z/3, 38C-13, WEHI-231, X16C, WEHI-279, and BCL₁. Only the WEHI-279 and BCL₁ lines produced detectable IL-6 constitutively, and the BCL₁ cells could be further induced by treatment with LPS. Of the remaining cell lines, only WEHI-231 and X16C could be stimulated with LPS to produce IL-6. To evaluate whether IL-6 could influence proliferation and Ig secretion by the cell lines, low cell density cultures were established in the presence of various doses of human rIL-6 and were assessed over time for levels of [³H]thymidine uptake and supernatant Ig. Under these conditions, IL-6 had no effect on either cell function.

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On the other hand, IL-6 overproduction in the brain leads to neurodegeneration [36,37]. IL-6 can be secreted by both immune (T-cells, B-cells, macrophages and microglia) [38–41] and non-immune cells (muscle cells, adipocytes, fibroblasts, endothelial cells, neurons) [32,42–45]. In contrast, the IL-6 receptor (IL-6R) is only found on a restricted subset of cell types, including hepatocytes [46], some leukocytes [47] and microglia [48,49] but not on oligodendrocytes [50] or astrocytes [48].
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C/EBPζ was originally identified as a gene induced upon DNA damage and growth arrest. It has been shown to be involved in the cellular response to endoplasmic reticulum stress. Because of sequence divergence from other C/EBP family members in its DNA-binding domain and its consequent inability to bind the C/EBP consensus-binding motif, C/EBPζ can act as a dominant negative inhibitor of other C/EBPs. C/EBP transactivators are essential to the expression of many proinflammatory cytokines and acute phase proteins, but a role for C/EBPζ in regulating their expression has not been described. We found that expression of C/EBPζ is induced in response to LPS treatment of B cells at both the mRNA and protein levels. Correlating with the highest levels of C/EBPζ expression at 48 h after LPS treatment, there is an increased association of C/EBPζ with C/EBPβ, and both the abundance of C/EBP DNA-binding species and IL-6 expression are downregulated. Furthermore, ectopic expression of C/EBPζ inhibited C/EBPβ-dependent IL-6 expression from both the endogenous IL-6 gene and an IL-6 promoter-reporter. These results suggest that C/EBPζ functions as negative regulator of IL-6 expression in B cells and that it contributes to the transitory expression of IL-6 that is observed after LPS treatment.
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Bupleuran 2IIc, a pectic polysaccharide isolated from the roots of Bupleurum falcatum L., was characterized as a T-cell-independent B cell mitogen, that activates, proliferates and differentiates B cells in vivo and in vitro (Immunology 97 (1999) 540). Studies were focused on elucidating the mechanism by which bupleuran 2IIc causes proliferation of B cells and expression of cell cycle regulatory proteins. B cells showed slower rates of entry into the S and G2/M phases of the cell cycle when stimulated with bupleuran 2IIc versus anti-IgM. However, the Stimulation Index continued up to two times longer with bupleuran 2IIc over anti-IgM. Although both bupleuran 2IIc and anti-IgM induced similar expressions of cell cycle regulatory proteins, cyclins D2, A, and B1, in B cells, those cells stimulated with bupleuran 2IIc appeared to sustain expressions of these protein for longer periods of time. Stimulation of B cells with bupleuran 2IIc induced phosphorylation of retinoblastoma protein, pRB, an important gene product regulating the restriction point, R, which is responsible for the transition from the G0/G1 to the S phases of the cell cycle. The results of this study demonstrate that both bupleuran 2IIc and anti-IgM interact with B cells, thus, leading to expressions of cell cycle regulatory proteins. However, the respective modes of binding and proximity of interactions with the B cell membrane may differ.
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Citation Excerpt :
In our previous studies, we found C/EBPβ to be predominantly in heterodimers with C/EBPγ in P388 B cells that are dependent upon transfected C/EBPβ expression for LPS induction of IL-6 and MCP-1 (Refs. 8 and 9; Fig. 2 A). We also found both C/EBPβ and C/EBPδ to be in heterodimers with C/EBPγ in WEHI-231, a B cell line that has been used in several studies of IL-6 expression (30-33). In these cells, LPS-induced IL-6 expression was associated with induction of C/EBPβ:γ and C/EBPδ:γ heterodimers (Fig. 2,B and C).
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^☆: This is publication No. 6199-IMM from the Department of Immunology, Research Institute of Scripps Clinic. This work was supported by U.S. Public Health Service Grants AR39178 (M.V.H.), AI07007, and Biomedical Research Support Grant RR05514.

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Interleukin-6 production by murine B cells and B cell lines☆

Abstract

Immunol. Today

Immunol Today

J. Immunol. Methods

Cell. Immunol

Anal. Biochem

Clin. Immunol. Immunopathol

Cell

Immunol. Today

Annu. Rev. Immunol

J. Immunol

J. Exp. Med

J. Exp. Med

Eur. J. Immunol

J. Immunol

J. Immunol

Nature (London)

Science

J. Exp. Med