Evidence for the effect of soluble uric acid in augmenting endoplasmic reticulum stress markers in human peripheral blood mononuclear cells

There is evidence regarding the association of hyperuricemia with inflammatory disorders. Hence, it has been of particular interest to dissect the exact role of alteration in uric acid (UA) levels in the context of inflammation. Recently, the endoplasmic reticulum (ER) stress pathway has come into the forefront as a possible mechanism linking hyperuricemia to inflammation. Here, we intended to examine the role of UA in the presence or absence of a second stimulus, LPS, in human peripheral blood mononuclear cells (PBMCs), and analyzed ROS production as well as expression of ER stress markers: GRP78 and CHOP, and inflammatory cytokines. PBMCs were isolated using Ficoll gradient centrifugation from healthy volunteers. Cell viability was measured by MTT assay. PBMCs were treated with an increasing concentration of soluble UA (0, 5, 12, and 20 mg/dl) for 20 h, followed by the addition of 100 ng/mL of LPS or vehicle for another 4 h. Real time-PCR was performed to investigate the mRNA expression of GRP78, CHOP, TNF-alpha, IL-1 beta, and IL-6, and western blot was used to investigate the protein levels of GRP78 and CHOP. Moreover, ELISA was used to evaluate the protein levels of TNF-alpha, IL-1 beta, and IL-6. Finally, intracellular ROS production was determined using fluorescent probes (DCFH-DA). High concentrations of UA either alone or combined with LPS increased the protein levels of GRP78 and CHOP. On the other hand, LPS alone increased the protein levels of GRP78 and CHOP. However, there was no significant difference between the mRNA expression of GRP78, CHOP, TNF-alpha, IL-1 beta, and IL-6 when PBMCs were treated with UA. High concentrations of UA augmented LPS-stimulated IL-1 beta transcript and protein levels as well as TNF-alpha protein levels in PBMC culture. Moreover, high concentrations of UA along with LPS significantly increased intracellular ROS production. It seems that a high concentration of UA not only induces the protein levels of ER stress markers in PBMCs but also augments the impact of LPS on the levels of pro-inflammatory markers and ROS production.