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

Abstract

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.

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

“…Irradiation of DCs increased expression of CD80 (Fig. 5c), as reported previously 6 . Expression was completely inhibited by addition of anti‐TNF‐α and anti‐CD154 mAbs (left panel), but 20 m m NAC did not affect irradiation‐induced CD80 expression (right panel).…”

“…In order to examine the antigen‐presenting ability of LPS‐stimulated B cells, 2 × 10 5 cells were incubated with 2 × 10 4 42‐6A cloned T cells 26 for 18 hr in 250 µl culture medium on a 96‐well culture plate in the presence of OVA or OVA 323−339 peptide at the indicated doses. To assess IL‐2 production, 50 µl of culture supernatant was then harvested from each well and added to CTLL‐2 cells, an IL‐2‐dependent T‐cell line, as previously described 5,6 , 12 . CTLL‐2 cells were pulsed with 0·25 mCi [ 3 H]thymidine during the last 6 hr of a 20 hr‐incubation.…”

“…Total cellular RNA was extracted using the single‐step guanidium–thiocyanate method, as described previously 6,28 . RNA samples were electrophoresed on a formaldehyde−1% agarose gel and were transferred onto a Zeta‐probe nylon membrane (Bio‐Rad Laboratories).…”

“…The former is given through the T‐cell antigen receptor (TCR) and the latter is provided by a costimulatory molecule, one of which is CD80 (B7‐1) 1–4 . We have previously shown that X‐irradiation of A20‐HL B lymphoma cells and dendritic cells (DCs) preferentially enhances expression of CD80 by inducing expression of tumour necrosis factor‐α (TNF‐α) and CD154 (CD40L), which results in enhanced antigen‐presenting ability 5,6 . Investigators recently reported that irradiation‐induced CD80 expression is not restricted to this lymphoma line, and is also observed in other tumour cell lines that have been maintained in vitro for a long period of time, such as MOPC315 plasmacytoma and P815 mastocytoma, 7–9 and in myeloid leukaemic cells freshly isolated from patients with acute myeloid leukemia 10 .…”

Suppression by Δ9-tetrahydrocannabinol of the primary immunoglobulin M response by human peripheral blood B cells is associated with impaired STAT3 activation

“…Pretreatment with Δ 9 -THC did not affect the upregulation of CD86 (Fig 2a and 2b), ICAM1 or CD69 (data not shown), but significantly suppressed the upregulation of surface CD80 (Fig 2c and 2d). As the induction of surface CD80 by CD40 ligation in B cells is primarily regulated at the level of transcription (Ishikawa et al 2002; Niu et al 2003), we next investigated whether Δ 9 -THC affected the levels of CD80 mRNA by RT-PCR. Pretreatment with Δ 9 -THC did not affect CD80 mRNA expression induced by CD40 plus cytokines in activated HPB B cells (Fig 3).…”