I) To further elucidate B cell isotype differentiation in humans we defined the conditions necessary for IgG sub-class-specific germline transcript (GLT) production in human peripheral blood cells. We found that induction of GLT for the various IgG subclasses fell into two patterns. Induction of Cgamma3 and Cgamma1, GLT, transcripts of genes in the first human duplication unit, generally required a proliferative stimulus (SAC) and was brought about by SAC plus IL-4 (Cgamma3 GLT) and SAC alone or SAC plus IL-2 (Cgamma1 GLT); in contrast, induction of Cgamma2 and Cgamma4 GLT, transcripts of genes in the second duplication unit, was induced accomplished with cytokines alone: IFN-gamma (Cgamma2 GLT) and IL-4 (Cgamma4 GLT), and was not augmented by addition of a proliferative stimulus. Finally, we found that IFN-gamma inhibited IL-4- induced Cgamma3 and Cgamma4 GLT (with or without SAC). These findings establish that the various human IgG subclasses manifest distinct requirements for the regulation of early steps in isotype differentiation. In addition, they suggest that human Cgamma genes exhibit patterns of regulation related to their respective gene duplication units. II) IL-10 has recently been shown to have an important influence on human B cell differentiation. To learn about the regulation of this cytokine we tested the hypothesis that its production by monocytes is under control of one of the inflammatory cytokines. In initial studies, we co-cultured purified human peripheral blood monocytes with a panel of cytokines including TNF-alpha, IL-1alpha, IL1-beta, IL-6, GM-CSF, TGF- beta and IFN-alpha, and then measured IL-10 mRNA production using a semi- quantitive reverse transcription-polymerase chain reaction (RT-PCR) technique. We found that TNF-alpha has a major effect on IL-10 mRNA production, inducing an 80-120-fold increase over baseline; in contrast, none of the other cytokines have more than a 2-3-fold effect. In later studies, we showed that the induction of IL-10 mRNA by TNF-alpha is dose- dependent and begins between 8-24 hr following the addition of TNF-alpha; this suggests that the increased IL-10 mRNA level is due to de nova mRNA synthesis rather than mRNA stabilization. Taken together, these results suggest that TNF-alpha plays a key role in the induction of IL-10 in human monocytes; as such, it induces a molecule that provides negative feedback to its sown production. In addition, these studies suggest that TNF-alpha, via its effect on IL-10, may affect overall levels of B cell proliferation.
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