Fig. 2.

Ligation of CD23 induces production of nitrites and TNF-α in 1321N1 astrocytoma cells. After CD23 induction by IFN-γ (1000 U/ml for 48 hr), cells were incubated for 48 hr with 10–20 μg/ml of anti-CD23 135 mAb to trigger the CD23 antigen, or with an isotype-matched negative control IgG1. Nitrite (A) and TNF-α (B) levels were determined in the supernatant 48 hr after the beginning of the treatment. Some of the experiments were performed in the presence of 1 mml-NAME (C). Results are expressed as the mean ± SEM in three independent experiments. *Significantly different compared with untreated cells (p < 0.01, two-tailed ttest). Significantly different from cultures treated with anti-CD23 mAb alone: †p < 0.05; ††p < 0.01, two-tailed t test. ND, Nondetectable. D, After CD23 ligation in IFN-γ-treated 1321N1 glial cells, total mRNA was extracted and assayed for iNOS mRNA expression by RT-PCR. HPRT amplification was used as a control. As a positive control some cells were treated with IFN-γ and LPS to induce iNOS. E, Western blot analysis of treated cells using mouse anti-human iNOS antiserum. In D andE, Control indicates untreated cells,IFN-γ indicates cells stimulated by IFN-γ, andIFN-γ+anti-CD23mAb indicates cells stimulated by IFN-γ and subsequently treated by a monoclonal antibody raised against CD23 or an isotype-matched immunoglobulin (IgG₁) as a negative control.