TNF-a and NO production in LPS-activated human mononuclear cells with disrupted-lipid rafts

MECHOUD M.A., FONT DE VALDEZ F, RODRIGUEZ A.V.

Mar del Plata, Argentina

Congreso; XL Reunión Anual SAIB; 2007

Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular ( SAIB )

TNF-a and NO production in LPS-activated human mononuclear cells with disrupted-lipid rafts Mechoud, M.A.; Font de Valdez, G.; Rodriguez, A.V. CERELA-CONICET. Chacabuco 145. 4000 Tucumán, Argentina. E-mail: mmechoud@cerela.org.ar Lipopolysaccharide (LPS) stimulates TNF-a production in immune cells through receptors localized in lipid rafts. Previously we founded that L. reuteri CRL 1098 inhibited TNF-a production in control (27%) and disrupted-lipid rafts cells (51%). Here we studied the effects of L. reuteri CRL 1098 on TNF-a and nitric oxide (NO) production of normal and disrupted-rafts cells activated with LPS.  Rafts of peripheral blood mononuclear cells were modified by cholesterol depletion with 10 mM methyl-b-clyclodextrin treatment. Control (no treated) and disrupted-lipid rafts cells were incubated with 100 ng/ml LPS at different times. TNF-a and NO production were measured by chemiluminiscence and Griess assays respectively. TNF-a value in the supernatant of 1x106 cells exposed to LPS was 514 pg/ml after 4 h incubation at 37ºC; 17% of inhibition in TNF-a levels was observed in disrupted-lipid cells in the same conditions. When L. reuteri or the supernatant of its culture was added to disrupted-lipid rafts cells 30% and 11% inhibition on TNF-a production was observed respectively. This effect was increased at 24 h of incubation. In presence of L. reuteri. NO synthesis is time dependent and was correlated with TNF-α produced by both LPS-activated control and disrupted-lipid rafts cells. Studies are currently in progress to further define the role of rafts in the mechanism involved in this response.