FIGURE 3.

HLMC proliferation in coculture with ASM is dependent on cell-cell contact and CADM1. A, In the presence of ASM-conditioned media, HLMC survived for 7 days but did not proliferate (red line). This survival effect was attenuated equally by neutralizing Abs to IL-6 (blue line) and SCF (green line), with a nonsignificant additive effect evident with both SCF and IL-6 neutralization (purple line), n = 6 experiments using three HLMC donors and six ASM donors. **, p < 0.01; and ***, p < 0.001 by one-way ANOVA. †, p < 0.05 by Tukey’s multiple comparison test comparing HLMC cultured in ASM-conditioned to addition of combined SCF and IL-6 neutralizing Abs. B, Separation of HLMC from ASM cells using a 0.4 μM Transwell insert inhibits their proliferation and survival. HLMC were cultured in media alone (black solid line), exogenous SCF/IL-6/FCS (light green solid line solid line), or in the presence of ASM without SCF/IL-6/FCS (red solid line). HLMC were also cocultured with ASM with separation by a 0.4 μM Transwell insert (T) to block physical contact between the two cell types in the presence and absence of isotype control Ab (red and gray dashed line respectively), neutralizing anti-IL6 Ab (blue dashed line), neutralizing anti-SCF Ab (green dashed line), or both anti-IL-6 and -SCF (purple dashed line), n = 5 experiments using three HLMC donors and five ASM donors. ***, p < 0.001 by one-way ANOVA. †, p < 0.05 by Tukey’s multiple comparison test comparing cocultured HLMC to the addition of Transwell inserts in the presence and absence of neutralizing Abs. C, In keeping with the Transwell experiments, addition of an adhesion-blocking Ab to CADM1 also prevented HLMC proliferation in coculture with ASM, suggesting that CADM1-dependent adhesion is an important component of the ASM-induced HLMC proliferation pathway, n = 5 experiments using three HLMC donors and five ASM donors. **, p < 0.01 by paired t test comparing isotype control cocultured HLMC with the anti-CADM1 Ab. D, HLMC-ASM coculture in the presence of neutralizing Abs to SCF, IL-6, and CADM1 revealed a minor additional effect over neutralization of each in isolation suggesting a cooperative effect between the three molecules, n = 6 experiments using three HLMC donors and six ASM donors. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 by paired t test comparing isotype control cocultured HLMC with anti-SCF/IL-6/CADM1-treated cells.