Immunostimulatory flagellin from Burkholderia pseudomallei effects on an increase in the intracellular calcium concentration and up-regulation of TNF-α by mononuclear cells

Using flow cytometry analysis, the flagellin of Burkholderia pseudomallei acts as a signalling inducer, and evokes an increase in the intracellular calcium ion concentration ([Ca²⁺]i) in human peripheral blood mononuclear cells (PBMC). The cells with increased [Ca²⁺]i segregate into the live monocyte gate and not into the live lymphocyte gates. The stimulated [Ca²⁺]i increase can be neutralized with antiflagellin antibodies. In the absence of [Ca²⁺], [Ca²⁺]i was increased rapidly in flagellin-treated cells compared to non-flagellin-treated cells only after the addition of 1 mM CaCl₂. Selective calcium antagonists were used to effectively block the [Ca²⁺]i signal, revealing that this signal was decreased by the addition of L-type calcium channel blockers (diltiazem, nifedipine and verapamil) and La²⁺ but was not changed by the addition of a T-type calcium channel blocker (flunarizine). It seemed that flagellin facilitates [Ca²⁺]i influx via a La²⁺ sensitive L-type cellular membrane channel. Furthermore, flagellin also acts as a TNF-α inducer in a time- and concentration- dependent manner when adhered mononuclear cells are treated with flagellin. This ability to induce TNF-α production was affected by the presence of [Ca²⁺] in the culture medium. It suggested that B. pseudomallei flagellin is an immuno-stimulatory molecule, causing an increase in [Ca ²⁺]i and an up-regulation of TNF-α, which may play an important role in the inflammation process.