Interleukin-4 (IL-4) in combination with IL-11 or IL-6 reverses the inhibitory effect of IL-3 on early B lymphocyte development.

We have previously described a two-step methylcellulose culture system in which individual primitive progenitors from 5-fluorouracil (5-FU)-treated mice were shown to have both myeloid and B lymphoid differentiation capacity. Highly enriched Lin-Sca+FU2d BM cells were cultured in methylcellulose in the presence of Steel factor (SF), interleukin-7 (IL-7), and pokeweed mitogen stimulated spleen cell conditioned medium (PWM-SCM). Primary mixed myeloid colonies were replated after 8-11 days into secondary cultures containing SF and IL-7, which supported the generation of B220+sIgM- pre-B cell colonies. A number of growth factors, including IL-6, IL-11, granulocyte colony-stimulating factor (G-CSF), and IL-12 were shown to be capable of substituting for PWM-SCM to support the B lymphoid potential of primary colonies. B lymphoid potential was not supported, however, in SF + IL-3 or in SF + IL-3 plus any single growth factor (IL-1 to -12, granulocyte-macrophage colony-stimulating factor [GM-CSF], G-CSF, erythropoietin [Epo], leukemia inhibitory factor [LIF], tumor necrosis factor-alpha [TNF-alpha], transforming growth factor-beta [TGF-beta], gamma interferon [IFN-gamma], or insulin-like growth factor-1 [IGF-1]), but was supported in SF + IL-3 + 5% PWM-SCM. Experiments were designed to identify the factor or factors in PWM-SCM that reverse the inhibitory effects of IL-3 on B lymphoid potential. By substituting various cytokine combinations for PWM-SCM, we determined that combinations of IL-4 + IL-6 or IL-4 + IL-11, but not IL-4 alone, can substitute for PWM-SCM to reverse the inhibitory effect of IL-3 on B lymphoid potential. Neutralizing antibodies to IL-4 completely eliminated the activity in PWM-SCM, but antibodies to IL-6 only partially inhibited the activity. IL-11 was not detected in PWM-SCM, and the activity co-purified with IL-4, but not with IL-6. Thus, IL-4 plus IL-6, IL-11, or one or more unidentified growth factors in PWM-SCM can reverse the inhibitory effects of IL-3 on early B lymphocyte development in culture.