Kinetics of Intracellular, Extracellular and Production of Pro-Inflammatory Cytokines in Lipopolysaccharide- Stimulated Human Peripheral Blood Mononuclear Cells

E Kumolosasi,E Salim,I Jantan,W Ahmad

doi:10.4314/tjpr.v13i4.8

Abstract

Purpose: To determine the detailed time course of intracellular, extracellular and production of proinflammatory cytokines (TNF-ƒα, IL-1β, IL-1&#945, IL-6 and IL-8) in normal human peripheral blood mononuclear cells (PBMC).Methods: PBMC were isolated from human whole blood, stimulated by 0.1 µ/ml lipopolysaccharide (LPS) and incubated at 37oC, 5% CO2. Samples were harvested at different time intervals (4, 8, 12, 16, 20 and 24 h) after stimulation. ELISA was employed for the measurement of the extracellular and intracellular cytokine levels of the samples.Results: The release of TNF-α, IL-6 and IL-8 on LPS-stimulated PBMC were significantly higher with concentrations in the range of 3161 ± 162.5 to 4027 ± 361.5 pg/ml (p < 0.001), 3921.5 ± 879.3 to 11628.3 ± 2647.3 pg/ml (p ≤ 0.030), and 4122.0 ± 382.9 to 5898.6±115.8 pg/ml (p ≤ 0.049), respectively compared to intracellular levels that were very low (TNF-α, 23.5 #177; 5.0 to 69.5 ± 13.8 pg/ml; IL-6, 22.5 ± 16.5 to 96.5 ± 9.6 pg/ml; and IL-8, 501.1 ± 221.0 to 1452.5 #177 415.7 pg/ml) and remained unchanged during 24 h. In contrast, both IL-1α and IL-1β 3946;were secreted gradually. Secretion and production of IL-6 was significantly higher at 8 h (9394.4 ± 846.3 pg/ml; p = 0.002) and at 20 h (11628.3 ± 2647.3 pg/ml; p = 0.014) than other cytokines.Conclusion: The differences in the characteristic kinetics of cytokines may be caused by different mechanisms of secretion and function. IL-1, TNF-945; and IL-8 play a role as proinflammatory cytokines, whereas IL-6 consecutively plays a dual role as pro-inflammatory cytokine and anti-inflammatory.Keywords: Pro-inflammatory cytokine, Tumor necrosis factor-α, Interleukin-1α, Interleukin-1β, Interleukin-6, Interleukin-8.

Highlights

Cytokines are low molecular weight proteins produced by cells in response to a variety of stimuli
Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), Interleukin-1α (IL-1α), Interleukin-6 (IL-6) and Interleukin-8 (IL-8) are pro-inflammatory cytokines which even though are dissimilar proteins transcribed from different genes, share important roles in acute inflammation [5]
The results show that the levels of TNF-α secreted in response to LPS stimulation were significantly higher

Summary

INTRODUCTION

Cytokines are low molecular weight proteins produced by cells in response to a variety of stimuli. The detailed time course of intracellular, extracellular and production of these proinflammatory cytokines was determined in normal human peripheral blood mononuclear cells (PBMC) induced by Salmonella enteritica LPS. Pelleted cells were washed with RPMI-1640 and lysed based on the procedure described by Hazuda et al [10] which used lysis buffer (40mM; 50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 1 mM dithiothreitol) plus 2 mM PMSF followed by sonication for 30 s, with modification by changing lysis buffer with water followed by gentle shaking for 10 s and left at room temperature for 1 h for measuring intracellular cytokines This modification was performed to avoid the use of chemicals which may influence the stability of the cytokines.

RESULTS

DISCUSSION

CONCLUSION