SUMMARY OF THESIS* * This thesis is available at the Library of the Instituto de Medicina Tropical de São Paulo
THE ROLE OF NATURAL KILLER CELLS IN THE EARLY PHASE OF Leishmania (Leishmania) amazonensis INFECTION IN MICE.
BALB/c and C57BL/6 mice were depleted in NK cells and/or in complement and infected subcutaneously with 5x107 stationary phase promastigotes of Leishmania (Leishmania) amazonensis and samples were taken at 3, 24, 48 hours and 7 days of infection. In NK-depleted mice the NK cytotoxic activity of the spleen cells was decreased and at 7 days of infection more parasites were found in the lesion. We observed in NK-depleted mice increase in the complement activity in the sera, increase and persistence in the C3 deposits at the parasite inoculation site. The IFN-g level, measured by capture ELISA was lower at 3 and 24 hours with recovery from 48 hours of infection in NK-depleted mice of both strains. The presence of IFN-g and IL-12 at the parasite inoculation site analyzed by immunohistochemistry showed that both cytokines were present but in higher amount in C57BL/6 than in BALB/c mice. There was more IFN-g and IL-12 at 24 hours and 7 days of infection in the NK-depleted than in non-depleted control mice. Two sub-populations of NK cells analyzed in the spleen cryosection by immunohistochemistry showed increase in the NK1.1 expression and a slight decrease in NK/5E6 expression on NK-depleted mice of both strains. The complement depletion leading a higher increase of the parasitism in NK-depleted animals lesion.
The data suggest that NK depletion by 90Sr leads to increase of the parasitism in the lesion. The pathogenetical mechanism involved in this process seems to be directly related to NK cells, since the depletion of complement leads to parasitism increase. The increase in NK1.1 expression, which produces mainly IL-4, may have role in the progression of the infection.
EFFECT OF INSULIN-LIKE GROWTH FACTOR (IGF)-I ON Leishmania in vitro AND ON THE EXPERIMENTAL CUTANEOUS LEISHMANIASIS
Insulin-like growth factor (IGF)-I, constitutively present in the skin, is likely to be one of the first host factor that the Leishmania parasites encounter after being transmitted by the insect vector and all along the course of infection. In the present study we have therefore scrutinized the in vitro effect of IGF-I on different species of Leishmania and in the experimental leishmaniasis.
IGF-I used at levels likely to be present in the skin (i.e.1-50 ng/ml) induced a proliferative response in metacyclic promastigotes from all tested species [Leishmania (Leishmania) chagasi, L. (L.) mexicana, L. (L.) amazonensis, L. (Viannia) panamensis] and in addition axenic amastigotes of L. (L.) mexicana in the presence of sub-optimal concentration of fetal calf serum (4%).
When phosphorylation pattern was analysed promastigotes and amastigotes of L. (L.) mexicana showed a rapid phosphorylation on proteins on tyrosine residues but also on other residues, however the predominant phosphorylation occurred on tyrosine residues. However the phosphorylation pattern was different in promastigotes and amastigotes. In promastigotes IGF-I induced more intense phosphorylation in 185 Kda protein that was shown to antigenically homologous to the substrate of the human insuline and IGF-I receptor (insulin receptor substrate-1 = IRS-1). The total phosphorylation pattern were similar upon IGF-I- or PMA (phorbol myristate acetate)- stimulation, the latter a well known activator of protein kinase C (PKC), suggesting that PKC pathway could also be involved in the IGF-I activation of Leishmanias.
IGF-I bound specifically with high affinity to a single site receptor on both promastigotes and amastigotes. Using the same strain we were able to make a direct comparison of characteristics of binding between promastigotes and amastigotes. The affinity was Kd= 0.4 nM in promastigotes and 1.0 nM in amastigotes, and the number of receptors/parasite 2 x 106 and 6 x 106, respectively. The partial characterization that we have done revealed a single chain glycosylated receptor with 65 KDa. This molecule was found to be antigenically related to IGF-I the receptor of human cells despite a smaller molecular mass.
In BALB/c mice inoculated with promastigotes of L (L.) panamensis pre-incubated with IGF-I, this factor induced a significant increase in the lesion size from 30 days post-infection. An increase in the polymorphonuclear neutrophil infiltrate was observed already at 3 hours of infection and subsequently an increase in the mononuclear cell infiltrate at 48 hours, 7 and 30 days PI with Leishmania parasites pre-incubated with IGF-I injection.
Challenge with Leishmania parasites pre- incubated with IGF-I resulted in higher degree of phagocytosis and parasites within macrophages throughout of infection period.
Our data suggest, considering the potential effect of IGF-I both on Leishmania and on host cells, that IGF-I constitutes one of the host derived factors that has important role in the pathogenesis of leishmaniasis.
Publication Dates
-
Publication in this collection
02 July 1999 -
Date of issue
Mar 1999
