Irradiation up-regulates CD80 expression through induction of tumour necrosis factor-alpha and CD40 ligand expression on B lymphoma cells

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Abstract
Previously, 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-alpha (TNF-alpha) and CD40 ligand (CD40L), and the amount of immunoprecipitable TNF-alpha and CD40L in cell lysates. The addition of anti-TNF-alpha 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-alpha and CD40L expression. Irradiation also increased the accumulation of CD80, TNF-alpha and CD40L mRNA. n-tosyl-l-phenylalanine chloromethyl ketone (TPCK), a nuclear factor (NF)-kappaB 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-alpha and CD40L, but also the up-regulation of CD80 on irradiated A20-HL cells. These results strongly suggested that irradiation induced TNF-alpha and CD40L expression, which then up-regulated CD80 mRNA and CD80 expression through activation of NF-kappaB transcription factor in A20-HL cells.