Leishmanial lipid suppresses tumor necrosis factor alpha, interleukin-1beta, and nitric oxide production by adherent synovial fluid mononuclear cells in rheumatoid arthritis patients and induces apoptosis through the mitochondrial-mediated pathway.
OBJECTIVE
Leishmanial lipid is a strong immunosuppressor of host cells. Inhibition of the inflammatory responses of synovial cells through induction of apoptosis is one of the main targets of therapeutic intervention in rheumatoid arthritis (RA). This study was undertaken to examine the antiinflammatory and apoptosis-inducing effects of leishmanial lipid on adherent synovial fluid mononuclear cells (SFMCs) in patients with RA.


METHODS
Lipid was extracted from a Leishmania donovani promastigote (MHO/IN/1978/UR6) by the Bligh and Dyer method. Nitric oxide (NO) was measured using the Griess reaction, and enzyme-linked immunosorbent assays for cytokines, NF-kappaB, and cytochrome c were performed. Levels of cytokines, inducible nitric oxide synthase, caspases, Bcl-2, Bax, t-Bid, and cytochrome c in the cell lysate and of NF-kappaB p65 in the nucleus were determined by Western blotting. Microscopic analysis, nuclear staining, DNA fragmentation assay, fluorescence-activated cell sorting, colorimetric assay for caspases, and fluorescent probe for measurement of mitochondrial membrane potential were used to study the leishmanial lipid-induced apoptotic pathway in SFMCs.


RESULTS
Leishmanial lipid inhibited the release of tumor necrosis factor alpha, interleukin-1beta, and NO in the culture, decreased their cytosolic protein levels, and decreased NF-kappaB p65 levels in SFMCs, in a dose-dependent manner. It had the reverse effect on interleukin-10 levels. Leishmanial lipid-induced apoptosis involved the activation of caspase 3, caspase 9, and Bax, the release of cytochrome c, the alteration of mitochondrial membrane potential, and the down-regulation of Bcl-2.


CONCLUSIONS
These results suggest that leishmanial lipid has strong antiinflammatory and apoptosis-inducing effects on SFMCs from patients with RA, and that apoptosis occurs via the mitochondrial pathway.
Majumdar
Kajal Nayan
KN
Indian Institute of Chemical Biology, Kolkata, India.
Banerjee
Aditi
A
Ratha
Jagnyeswar
J
Mandal
Mriganka
M
Sarkar
Rathindra Nath
RN
Saha
Krishna Das
KD
D000328:Adult; D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D016753:Interleukin-10 / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism*; D007893:Leishmania donovani / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008055:Lipids / Q000494:pharmacology*; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000502:physiology; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D013582:Synovial Fluid / Q000166:cytology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D053583:Interleukin-1beta; D008055:Lipids; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D009569:Nitric Oxide; D053148:Caspase 3; D053453:Caspase 9