We tested several monokines and muramyl dipeptide (MDP) to determine whether they induce the L-arginine-dependent effector mechanism in cultured murine macrophages. Recombinant interferon-gamma (rIFN-gamma) and recombinant tumor necrosis factor (rTNF) synergize to induce nitrite (NO2-) and nitrate (NO3-) synthesis from L-arginine as well as to cause inhibition of the iron-dependent enzyme aconitase in macrophages. Unlike rTNF, recombinant interleukin 1 (rIL 1) and rIL 6/B cell stimulatory factor 2 (rIL 6/BSF-2) did not act as cofactors when added to macrophages in the presence of rIFN-gamma. rIFN-gamma plus MDP induced the L-arginine-dependent effector mechanism in murine macrophages. However, induction by rIFN-gamma plus MDP was inhibited by anti-rTNF antibodies which suppressed both NO2-/NO3- synthesis and aconitase inhibition. This result indicates that endogenously produced TNF is involved in the induction of the L-arginine-dependent effector mechanism when MDP is the co-stimulant with rIFN-gamma. In contrast, anti-rTNF antibodies did not fully suppress the effect of combining rIFN-gamma and lipopolysaccharide, suggesting that, in this case, activation of the L-arginine-dependent effector pathway may involve more than induction of TNF synthesis by the macrophages. These results provide information, at a biochemical level, on a mechanism through which combination of IFN-gamma and TNF can modulate macrophage functions involved in the control of cell proliferation.

Biosynthesis of nitric oxide (NO) from L‐arginine modulates activity of iron‐dependent enzymes, including mitochondrial acontiase, an [Fe‐S] protein. We examined the effect of NO on the activity of iron regulatory factor (IRF), a cytoplasmic protein which modulates both ferritin mRNA translation and transferrin receptor mRNA stability by binding to specific mRNA sequences called iron responsive elements (IREs). Murine macrophages were activated with interferon‐gamma and lipopolysaccharide to induce NO synthase activity and cultured in the presence or absence of NG‐substituted analogues of L‐arginine which served as selective inhibitors of NO synthesis. Measurement of the nitrite concentration in the culture medium was taken as an index of NO production. Mitochondria‐free cytosols were then prepared and aconitase activity as well as IRE binding activity and induction of IRE binding activity were correlated and depended on NO synthesis after IFN‐gamma and/or LPS stimulation. Authentic NO gas as well as the NO‐generating compound 3‐morpholinosydnonimine (SIN‐1) also conversely modulated aconitase and IRE binding activities of purified recombinant IRF. These results provide evidence that endogenously produced NO may modulate the post‐transcriptional regulation of genes involved in iron homeostasis and support the hypothesis that the [Fe‐S] cluster of IRF mediates iron‐dependent regulation.

We have carried out a longitudinal study of interferon (IFN) and tumor necrosis factor (TNF) using a whole-blood mitogen stimulation assay in 20 multiple sclerosis (MS) patients and in a healthy control group. We set up individual profiles and the results were quite constant for each individual, both in healthy donors and in the patients in remission. Before exacerbations, however, we found an increase of IFN-gamma and TNF production preceding clinical symptoms by a maximum of 2 weeks. In benign cases, the increase disappeared rapidly, even before the appearance of symptoms, whereas we found sequelae whenever the increase persisted during weeks. In chronic progressive patients, we frequently found intervening increases. It may be that IFN-gamma and TNF trigger off exacerbations at a very early stage and that these cytokines may also play a role in maintaining disease in chronic progressive and invalidating forms.

We investigated the molecular basis of the synergistic induction by interferon-␥ (IFN-␥)/tumor necrosis factor-␣ (TNF-␣) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-␥ and/or TNF-␣ action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between ؊73 and ؊36, which is the minimal element inducible by LPS or TNF-␣; 2) an element located between ؊181 and ؊73, which appeared to regulate the response to IFN-␥ and TNF-␣ negatively; and 3) a distal element upstream of ؊224, which was inducible by IFN-␥ alone. LPS signaling was found to involve NFB activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-␥ and TNF-␣, in monocytic cells, involved cooperation between the IRF-1 and NFB p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-␥.

SUMMARY Inoculation of Trypanosoma cruzi, Tulahuén strain, into C57BL/6 and BALB/c mice led to an acute infection characterized by marked parasitaemia, myocardial inflammation and thymocyte depletion. While C57BL/6 mice showed a progressive and lethal disease, BALB/c mice partly recovered. To characterize these murine models more effectively, we studied the parasite burden, serum levels of major infection outcome‐related cytokines, the in vitro features of T. cruzi infection in peritoneal macrophages and the immunophenotype of thymic cells. The greater disease severity of T. cruzi‐infected C57BL/6 mice was not linked to an increased parasite load, as parasitaemia, myocardial parasite nests and amastigote counts in peritoneal macrophages were not different from those in BALB/c mice. Cortical thymocyte loss was accompanied by the presence of apoptotic bodies and fragmented nuclear DNA, whereas fluorocytometric analysis at 17 days postinfection (p.i.) revealed a more pronounced loss of CD4+ CD8+ cells in C57BL/6 mice. This group displayed higher levels of TNF‐α on days 14 and 21 p.i., in the presence of lower IL‐1β and IL‐10 concentrations by days 14 and 21, and days 7 and 14 p.i., respectively. Day‐21 evaluation showed higher concentrations of nitrate and TNF‐α soluble receptors in C57BL/6 mice with no differences in IFN‐γ levels, with respect to the BALB/c group. Increased morbidity of C57BL/6 T. cruzi‐infected mice does not seem to result from an aggravated infection but from an unbalanced relationship between pro‐ and anti‐inflammatory mediators.

Type I IFNs (IFN-α/β) have only recently gained considerable attention as immunomodulators in nonviral infectious diseases. IFN-β has been shown to protect, in a NO-dependent manner, against murine Old World leishmaniasis caused by Leishmania major, but data in New World leishmaniasis are lacking. We found that IFN-β dose-dependently increases parasite burden in Leishmania amazonensis- as well as Leishmania braziliensis-infected human macrophages, independent of endogenous or exogenous NO. However, IFN-β significantly reduced superoxide release in Leishmania-infected as well as uninfected human macrophages. This decrease in superoxide production was paralleled by a significant IFN-β-mediated increase in superoxide dismutase 1 (SOD1) protein levels. Additionally, IFN-β inhibition of leishmanicidal activity was mimicked by SOD1 and antagonized by either pharmacological or small interfering RNA-mediated inhibition of SOD1. Finally, pronounced SOD1 expression in situ was demonstrated in biopsies from New World cutaneous leishmaniasis patients. These findings reveal a hitherto unknown IFN-β/SOD1 axis in Leishmania infection and suggest that inhibition of SOD-associated pathways could serve as strategy in the treatment of L. amazonensis as well as L. braziliensis infection, major human pathogens.

Four human cell lines derived from Ewing's sarcoma, EW-7, EW-1, COH and ORS, were investigated to establish the eects of human recombinant interferon-a2a and human recombinant interferon-b on cell proliferation and apoptosis. All four cell lines were much more sensitive to the antiproliferative eects of IFN-b than of IFN-a. Analysis of the early signals triggered by IFN-a and IFN-b demonstrated that the two IFNs were similarly eective in inducing tyrosine phosphorylation of the Jak-1 and Tyk-2 kinases and the transcription factors Stat-1 and Stat-2. Interestingly, an additional rapid phosphorylation of Stat-1 on serine was observed after IFN-b treatment, with concomitant activation of p38 mitogen-activated protein kinase. In these cells, Stat-1 Ser727 phosphorylation in response to IFN-b was found to be impaired by p38 MAPkinase inhibitor (SB203580). IFN-b induced the formation of the Interferon Stimulated Gene Factor 3 complex more eciently than IFN-a, as well as sustained induction of IRF-1, which may account for its greater induction of 2'5'oligo(A)synthetase and greater inhibition of cell proliferation. IFN-b, but not IFN-a, induced apoptosis in wild-type p53 EW-7 and COH cell lines, but not in the mutated p53 EW-1 or ORS cell lines. The apoptosis induced by IFN-b in EW-7 and COH cell lines appeared to be mediated by IRF-1 and involved the activation of caspase-7. Ectopic expression of IRF-1 induced apoptosis in all four cell lines which correlated with the activation of caspase-7 and with the downregulation of the Bcl-2 oncoprotein, as observed for IFN-b-induced apoptosis in parental EW-7 and COH cell lines. Oncogene (2000) 19, 3372 ± 3383.

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