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Glycosaminoglycans (GAGs), especially heparin and heparan sulfate (HS), modulate the functions of numerous cytokines. The aims of this multidisciplinary research were to characterize heparin binding to interleukin-12 (IL-12) and determine the mechanism(s) by which heparin influences IL-12 bioactivity. Heparin and HS were found to bind human IL-12 (hIL-12) with low micromolar affinity and increase hIL-12 bioactivity by more than 6-fold. Conversely, other GAGs did not demonstrate significant binding, nor did their addition affect hIL-12 bioactivity. Biophysical studies demonstrated that heparin induced only minor conformational changes while size-exclusion chromatography and small angle X-ray scattering studies indicated that heparin induced dimerization of hIL-12. Heparin modestly protected hIL-12 from proteolytic degradation, however, this was not a likely mechanism for increased cytokine activity in vitro. Flow cytometry studies revealed that heparin increased the amount of hIL-12 bound to cell surfaces. Heparin also facilitated hIL-12 binding and signaling in cells in which both hIL-12 receptor subunits were functionally deleted. Results of this study demonstrate a new role for heparin in modulating the biological activity of IL-12.

Interleukin-12 (IL-12) is a heterodimeric molecule composed of p35 and p40 subunits. Analyses in vitro have defined IL-12 as an important factor for the differentiation of naive T cells into T-helper type 1 CD4+ lymphocytes secreting interferon-gamma (refs 1, 2). Similarly, numerous studies have concluded that IL-12 is essential for T-cell-dependent immune and inflammatory responses in vivo, primarily through the use of IL-12 p40 gene-targeted mice and neutralizing antibodies against p40. The cytokine IL-23, which comprises the p40 subunit of IL-12 but a different p19 subunit, is produced predominantly by macrophages and dendritic cells, and shows activity on memory T cells. Evidence from studies of IL-23 receptor expression and IL-23 overexpression in transgenic mice suggest, however, that IL-23 may also affect macrophage function directly. Here we show, by using gene-targeted mice lacking only IL-23 and cytokine replacement studies, that the perceived central role for IL-12 in autoimmune inflammation, specifically in the brain, has been misinterpreted and that IL-23, and not IL-12, is the critical factor in this response. In addition, we show that IL-23, unlike IL-12, acts more broadly as an end-stage effector cytokine through direct actions on macrophages.

Interleukin-12 (IL-12) is a heterodimeric cytokine that is central to the development of T helper 1-dependent cellular immunity. Although this cytokine has potential therapeutic application as an antineoplastic agent, the systemic infusion of IL-12 has led to toxic fatalities; hence, restriction of expression of IL-12 to the microenvironment of target tumor cells has obvious appeal. In this study, we examined whether tumor cells that were liposome-transfected with IL-12 could enhance the induction of cytolytic lymphocyte immunity to the native tumor. The plasmid expression vector that we used has several useful features including replication to high copy number as an episome and a polycistronic message enabling the production of both the p35 and p40 subunits of IL-12 without alternative splicing; up to 3 ng/mL/10(6)/48 hours of IL-12 was produced following transfection. Tumor cells transfected with IL-12 were superior to untransfected cells in the induction of lymphocyte-mediated cytolysis. IL-12 transfectants induced a heterogeneous population of natural killer, lymphokine activated killer, and cytolytic T lymphocytes, the latter of which exhibited tumor-specific activity. Our studies suggest that liposome-mediated transfection of tumor cells with an episomal, high copy number plasmid vector expressing both IL-12 subunits is a promising approach to cancer vaccination, a strategy that could be implemented ex vivo in treating malignancies such as metastatic ovarian cancer.

Cell-mediated immunity (CMI) in neonates is relatively deficient when compared with adults. Defects in cytokine production and/or regulation may contribute to heightened susceptibility to infection by intracellular pathogens. The heterodimeric cytokine IL-12 is a key regulator of CMI and inducer of interferon-gamma (IFN-gamma) production. We report here that umbilical cord blood-derived mononuclear cells (MNC) are capable of producing IL-12 (p40 subunit, measured by RIA, and IL-12 p70 heterodimer, by ELISA) at levels comparable to or greater than adult peripheral blood MNC, after stimulation with heat-killed Staphylococcus aureus in 18-h cultures. As in adult MNC, S. aureus induced IL-12 p40 mRNA accumulation in cord blood MNC. IFN-gamma was also produced in the S. aureus-stimulated cultures, in an IL-12-dependent manner, but cord blood MNC produced 5-fold lower levels of IFN-gamma compared with adult MNC (p < 0.05). Preincubation with IL-10 inhibited IL-12 p40 production by cord blood and adult peripheral blood MNC in a dose-dependent fashion, whereas neutralization of endogenous IL-10 enhanced IL-12 and IFN-gamma levels. The results demonstrate that the relative CMI deficiency in neonates is not due to an intrinsic defect in the capacity of neonatal MNC to produce IL-12. The underlying factors responsible for diminished IFN-gamma production are not known, but may lie in the balance of stimulatory and inhibitory signals delivered to the IFN-gamma secreting cells along with IL-12, or may relate more to the absence of memory T cells among cord blood MNC.