Cross-linking CD15 induced TNF-α release. Human peripheral blood mononuclear leukocytes prepared by ficoll gradient centrifugation (2 × 10⁶ cells) were incubated with three anti-CD15 IgM MoAbs (PM81, 80H5, UN-172) at a concentration of 10 μg/mL for 20 minutes at 4°C. Cells were then cross-linked by the addition of goat anti-IgM F(ab) ′₂ and allowed to incubate for 6 hours at 37°C. TNF-α released into the culture supernatant was then determined by ELISA. A representative of six separate experiments is shown. Cross-linking mononuclear leukocytes with anti-CD31 (MoAb hec-7), anti-CD11/CD18 (MoAb IB4), or anti-HLA (MoAb W6/32) for 6 hours was used for comparison. To determine the cell source of TNF-α, monocyte-depleted peripheral blood mononuclear cells (<95% lymphocytes) were shown not to screte TNF, either under basal conditions (labeled control) or after CD15 cross-linked for 6 hours at 37°C (last column). ⁺ = Significantly different from control (P < .05).