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In IL-5 transgenic mice (C3H/HeN-TgN(IL-5)-Imeg), in which 50% of peripheral blood leukocytes are eosinophils, the development of infection by Leishmania amazonensis was clearly suppressed. To determine mechanistically how this protozoan parasite is killed, we performed in vitro killing experiments. Either IL-4 or IFN-gamma effectively stimulated eosinophils to kill Leishmania amazonensis promastigotes, and most of the killing was inhibited by catalase but not by the NO inhibitor L-N5-(1-iminoethyl)-ornithine, suggesting that hydrogen peroxide is responsible for the killing of L. amazonensis by eosinophils. There was no significant degranulation of eosinophils in the culture, because eosinophil peroxidase was not detected in culture supernatants when L. amazonensis promastigotes were killed by activated eosinophils. Such resistance was also observed in BALB/c mice, which are highly susceptible to L. amazonensis. Expression plasmids for IL-4, IL-5, and IFN-gamma were transferred into muscle by electroporation in vivo starting 1 week before infection. Expression plasmid for IL-5 was most effective in slowing the development of infection among three expression plasmids. Expression plasmid for IL-4 was slightly effective and that for IFN-gamma had no effect on the progress of disease. These results suggest that IL-5 gene transfer into muscle by electroporation is useful as a supplementary protection method against L. amazonensis infection.

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Stimulation of macrophages with IgG2 immune complexes induced dose-dependently the O2- generation and the release of arachidonic acid and its metabolites. This Fc gamma R-mediated O2- generation was inhibited by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB), in parallel to the dose-dependent inhibition of arachidonic acid release. The main arachidonic acid metabolites released were shown to be prostaglandin E2 and thromboxane B2 and blocking of the production of these metabolites by indomethacin did not inhibit the O2- generation. Inhibition of the Fc gamma R-mediated O2- generation and the arachidonic acid release by the C-kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), was less intense than by 4-pBPB. These results support the previously proposed hypothesis that arachidonic acid acts as an intracellular activator of the Fc gamma R-mediated O2- generation in macrophages. Although the C-kinase activation may also contribute to the activation of the O2--generating system, arachidonic acid release appears to play a major role in Fc gamma R-mediated O2- generation. In contrast, activation of C-kinase seems to be contributing mainly in the induction of both the arachidonic acid release and O2- generation by 12-o-tetradecanoylphorbol 13-acetate (TPA). Furthermore, suboptimal concentrations of TPA and arachidonate were found to act synergistically to stimulate O2- generation and the inhibition study suggested a positive synergism between C-kinase and arachidonic acid release to induce O2- generation. This synergistic action may have general importance in receptor-mediated O2- generation.

In all vertebrate species, the homeobox gene goosecoid serves as a marker of the Spemann organizer tissue. One function of the organizer is the induction of neural tissue. To investigate the role of goosecoid in neuronal differentiation of mammalian cells, we have introduced goosecoid into PC12 cells. Expression of goosecoid resulted in reduced cell proliferation and enhanced neurite outgrowth in response to NGF. Expression of goosecoid led to a decrease in the percentage of S-phase cells and to upregulation of the expression of the neuron-specific markers MAP-1b and neurofilament-L. Analysis of goosecoid mutants revealed that these effects were independent of either DNA binding or homodimerization of Goosecoid. Coexpression of the N-terminal portion of the ets transcription factor PU.1, a protein that can bind to Goosecoid, repressed neurite outgrowth and rescued the proliferation of PC12 cultures. In contrast, expression of the bHLH transcription factor HES-1 repressed goosecoid-mediated neurite outgrowth without changing the proportion of S-phase cells. These results suggest that goosecoid is involved in neuronal differentiation in two ways, by slowing the cell cycle and stimulating neurite outgrowth, and that these two events are separately regulated.

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