Gene expression and intracellular NF-κB activation after HMGB1 and LPS stimuli in neutrophils from septic patients
© BioMed Central Ltd 2005
Published: 9 June 2005
Neutrophils play a major role in sepsis-induced organ dysfunction, especially in the lung. HMGB1 has emerged as a late cytokine and is implicated in the perpetuation of inflammatory stimulus and organ dysfunction development as well. There are limited data about neutrophil response patterns to HMGB1 in septic patients, and whether those patterns could be different from those following LPS exposure.
To evaluate the differences of gene expression and activation of NF-κB, Akt, and p38MAPK in blood neutrophils from septic patients exposed to HMGB1 and LPS; and to compare response patterns between blood neutrophils from patients and healthy volunteers.
Twenty-two sepsis-induced acute lung injury patients and 34 healthy volunteers were enrolled in this study. The primary clinical variables collected were the 28-day survival and the presence of shock at ICU admission. Peripheral blood was obtained and neutrophils were isolated by plasma–percoll gradients after dextran sedimentation of erythrocytes. Neutrophils were resuspended in RPMI and cultured with or without 1000 ng/ml rHMGB1 or with or without 100 ng/ml LPS for 15, 30, and 60 min. The electrophoretic mobility shift assay technique was used to measure the NF-κB translocation, while western blot analysis was used to determine Akt phosphorylation and an ELISA was used to determine p38MAPK phosphorylation. Microarray analysis was used to evaluate the neutrophil gene expression in unstimulated neutrophils and after either HMGB1 stimulus or LPS stimulus. P < 0.05 was considered significant.
Although with some similarities, HMGB1 and LPS induced distinct pattern of gene expression in neutrophils from septic patients. Both stimuli were able to increase intracellular activation and this activation was similar to that found in neutrophils from volunteers, showing that even after sepsis stimulus the neutrophil keeps its ability to respond to a second hit.