Induction of multiple cytokine gene expression and IRF-1 mRNA by flavone acetic acid in a murine macrophage cell line.

Flavone-8-acetic (FAA) acid is a potential chemotherapeutic agent that has demonstrated strong immunomodulatory activity in murine model systems. The immunomodulatory activity of this drug in murine systems has been linked to its ability to rapidly induce cytokine gene expression in vivo and in mouse splenocytes ex vivo. We have now developed a tissue culture model for studying the molecular basis of induction of cytokine expression by FAA. Using the mouse macrophage cell line, ANA-1, we can demonstrate the direct induction of interferon beta (IFN beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), and interferon response factor-1 (IRF-1) mRNA expression following treatment with FAA. Furthermore, the induction of the IFN beta mRNA can occur in the absence of new protein synthesis. Nuclear run-on experiments indicate that at least part of the induction of IFN beta, IL-6, and TNF alpha mRNA occurs at the transcriptional level while the increase in IRF-1 mRNA appears largely post-transcriptional or due to the production of IFN beta protein. Additionally, experiments using agents that interfere with second messengers demonstrate that activation of the protein kinase C pathway is possibly involved in FAA gene induction. The use of this tissue culture model system should lead to a more complete understanding of the mechanisms involved in FAA-induced gene expression and help determine why this drug is inactive on human cells.

Inducible accessory function of a macrophage cell line.

Costimulatory molecules in addition to occupancy of the T-cell antigen receptor, are required to induce T-cell proliferation. Previous work suggested that membrane molecules responsible for costimulatory activity were not constitutively expressed on the antigen presenting cell (APC) surface. In the present study, we have identified a cloned macrophage cell line (FLJ2) with inducible APC function. The unactivated FLJ2 line could not induce T-cell proliferation. FLJ2 could present alloantigen, and stimulate proliferation of either a T-cell clone or normal resting T cells following activation with IFN gamma or unexpectedly with lipopolysaccharide (LPS)-Activated FLJ2 cells could be fixed and APC function was preserved. The relevant inducible molecules required for APC function appeared distinct from Ia and IL1. The expression of ICAM-1 and LFA-1 was increased during activation and anti-LFA-1 antibody blocked APC function. This suggests that one important feature of the activation process may be improvement of cellular adhesion.

Growth of immature (double negative) rat thymocytes in the presence of IL-1.

It is unclear which growth factors, if any, are involved in the growth and differentiation of immature T cells in the thymus. Because IL-1 has been previously implicated in thymocyte proliferation, we examined the effects of IL-1 on precursor thymocytes, the CD4-CD8- or double-negative (DN) cells. We show that IL-1 (together with Con A) is a growth factor for DN, TCR- alpha beta-cells in vitro. After 5 days of culture in IL-1 and Con A, a number of phenotypic changes were observed and two subsets of DN cells were distinguished. One subset expressed full length TCR-alpha and -beta mRNA and surface alpha beta TCR, the other expressed low levels of full length TCR-gamma together with high levels of full length TCR-delta mRNA. Thus, DN cells are induced by IL-1 and Con A to proliferate and express TCR without parallel acquisition of CD4 or CD8 markers. These data suggest that IL-1 drives early steps of intrathymic T cell differentiation.