SUMMARYPreviously, we reported that 100 Gy X-ray irradiation followed by 24 hr incubation up-regulates CD80 expression in murine B lymphoma cells, A20-2J. In the present study, we analysed the underlying mechanisms of such up-regulation using A20-HL cells derived from A20-2J cells. Irradiation of A20-HL cells with 100 Gy enhanced CD80 expression. Incubation of untreated A20-HL cells with those 100 Gy irradiated induced up-regulation of CD80 expression. Irradiation of A20-HL cells also up-regulated the expression of tumour necrosis factor-a (TNF-a) and CD40 ligand (CD40L), and the amount of immunoprecipitable TNF-a and CD40L in cell lysates. The addition of anti-TNF-a or anti-CD40L monoclonal antibody (mAb) to the incubation of irradiated A20-HL cells partially inhibited up-regulation of CD80 expression, and the addition of both antibodies together almost completely inhibited the up-regulation, suggesting that irradiation up-regulated the CD80 expression through the induction of TNF-a and CD40L expression. Irradiation also increased the accumulation of CD80, TNF-a and CD40L mRNA. n-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a nuclear factor (NF)-kB inhibitor, markedly decreased irradiation-induced accumulation of CD80 mRNA and CD80 expression. FK506, a calcineurin inhibitor, and nifedipine, a calcium channel inhibitor, inhibited not only the expression of TNF-a and CD40L, but also the up-regulation of CD80 on irradiated A20-HL cells. These results strongly suggested that irradiation induced TNF-a and CD40L expression, which then up-regulated CD80 mRNA and CD80 expression through activation of NF-kB transcription factor in A20-HL cells.
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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