Abstract
To observe the pro- and anti-inflammatory cytokine (TNF-α, IL-4, IL-6, IL-10) secretion from cultivated peripheral blood mononuclear cells (PBMCs) for 48h. Blood samples were obtained from 42 women patients of Nadezhda Women’s Health Hospital after signing written informed consent. For PBMC isolation, 9 ml of heparinised whole blood from each patient were processed by density gradient centrifugation using Pancoll (P04-60100, Pan-Biotech) for 25 minutes at 400G. The buffy coat of PBMCs were collected and washed with phosphate buffer saline. A total of 5x106 /ml cells were cultured in 0.5 ml RPMI medium (R-8758, Sigma) supplemented with 1% HSA (GHSA-125, LifeGlobal) for 48 hours at 37ºC. Concentrations of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-4 (IL-4) and interleukin-10 (IL-10) in the PBMC media were measured by sandwich enzyme-linked immunosorbent assay (ELISA) (CSB-E04638h, CSB-E04740h, CSB-E04633h and CSB E04593h, Cusabio Technology, respectively) according to the manufacturers’ instructions. Results are presented as mean ±SD and range. Descriptive statistics and one-way ANOVA with post hoc LSD were performed with SPSS Software ver. 21. P<0.05 was considered significant. No cytokines were detected in the clear RPMI media control. The presence of IL-4, IL-6, IL-10 and TNF-α was confirmed in all PBMC samples. The proinflammatory cytokines (IL-6 and TNF-α) concentrations in the PBMC culture media after 48h of cultivation were 90.48±89.1 pg/ml (range: 25.05 – 266.43) and 510.36±66.81 pg/ml (range: 400.59 – 565.10), respectively. The mean concentrations of the anti-inflammatory cytokines (IL-4 and IL-10) secreted from the PBMC at the 48h of cultivation were 438.82±130.27 pg/ml (range: 227.53 - 599.19) and 4667.33±3505.7 pg/ml (range: 1808.31 – 9964.99), respectively. Additionally the secretion of IL-10 was significantly higher in comparison to the other analysed cytokines (p<0.001). Results from this study showed that PBMCs secrete detectable amounts of pro- and anti-inflammatory cytokines in the culture media. Furthermore the PBMC culture supernatant had significantly higher amount of IL-10 in comparison to the other studied cytokines (IL-4, IL-6 and TNF- α).
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