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Macrophage protein kinase C: its role in modulating membrane microviscosity and superoxide in leishmanial infection
P Chakraborty1, D Ghosh, M K Basu
1Biomembrane Division, Indian Institute of Chemical Biology, Raja S.C. Mullick Road, Jadavpur, Calcutta-700032, India.
Abstract:
Pretreatment of macrophages with, an agonist of PKC, showed diverse effects on degradation and survival of two virulent strains of Leishmania donovani promastigotes. Treatment of macrophages with PMA for 45 min at 37 degrees C generated significant amounts of superoxide anions and reduced the parasite burden of macrophages by up to 48 and 43% when AG83 and GE-1 strains were used for infection. Staurosporine, an inhibitor of PKC, inhibited PMA-dependent killing of the parasites, while tyrphostin AG 126, an inhibitor of protein tyrosine kinase, showed very little effect. Depletion of PKC by prolonged incubation with PMA drastically reduced the superoxide anion generation and increased the uptake and multiplication of the parasites. Finally, to understand the mechanism of higher uptake of the parasites by PKC-depleted macrophages, membrane microviscosity was measured by fluorescence depolarization. Membrane microviscosity was found to be approximately 40% lower in PKC-depleted macrophages than in normal macrophages, indicating the role of membrane fluidity in the infection process. Together, these data suggest PKC activation, superoxide generation, and membrane fluidity are essential factors in the efficient regulation of leishmanial infection.
Insights
Protein kinase C (PKC) activation in macrophages enhances superoxide anion generation, reducing Leishmania donovani parasite burden. PKC depletion increases parasite uptake and multiplication, highlighting PKC
Area of Science:
- Immunology
- Cell Biology
- Parasitology
Background:
- Leishmania donovani infects macrophages, causing leishmaniasis.
- The role of protein kinase C (PKC) in macrophage defense against L. donovani is not fully understood.
Purpose of the Study:
- To investigate the role of PKC activation in macrophage-mediated killing of L. donovani.
- To elucidate the mechanisms underlying PKC's influence on parasite burden and macrophage function.
Main Methods:
- Macrophage pretreatment with phorbol 12-myristate 13-acetate (PMA), a PKC agonist.
- Measurement of superoxide anion generation.
- Assessment of Leishmania parasite burden (AG83 and GE-1 strains).
- Inhibition studies using staurosporine (PKC inhibitor) and tyrphostin AG 126 (tyrosine kinase inhibitor).
- PKC depletion studies and measurement of membrane microviscosity via fluorescence depolarization.
Main Results:
- PMA treatment increased superoxide anion generation, reducing parasite burden by up to 48%.
- PKC inhibition (staurosporine) blocked PMA-dependent parasite killing.
- PKC depletion reduced superoxide generation and increased parasite uptake and multiplication.
- PKC depletion decreased macrophage membrane microviscosity, indicating increased fluidity.
Conclusions:
- PKC activation is crucial for macrophage defense against L. donovani.
- Superoxide anion generation and altered membrane fluidity are key mechanisms regulated by PKC in leishmanial infection.
- PKC, superoxide production, and membrane fluidity are essential for controlling leishmanial infection.