Leukocyte phosphodiesterase expression after lipopolysaccharide and during sepsis and its relationship with HLA-DR expression.

Phosphodiesterases (PDEs) may modulate inflammatory pathways, but PDE expression is poorly documented in humans with sepsis. Using quantitative PCR on whole blood leukocytes, we characterized PDE mRNA expression in healthy volunteers (n = 20), healthy volunteers given lipopolysaccharide (LPS; n = 18), and critically ill patients with (n = 20) and without (n = 20) sepsis. PDE4B protein expression was also studied in magnetic-activated cell sorting (MACS)-isolated CD15+ neutrophils (from 7 healthy volunteers, 5 patients without and 5 with sepsis). We studied relationships between PDE expression, HLA-DR (mRNA and expression on CD14+ monocytes), tumor necrosis factor (TNF)-α, and interleukin (IL)-10 levels. LPS administration in volunteers was associated with increases in PDE4B and PDE4D and decreases in PDE4A and PDE7A mRNAs. The observed global down-regulation of the HLA-DR complex was correlated with PDE7A. Critically ill patients had lower TNF-α/IL-10 mRNA ratios than the volunteers had and global down-regulation of the HLA-DR complex. Septic patients had persistently lower mRNA levels of PDE7A, PDE4A, and 4B (also at a protein level) and decreasing levels of PDE4D over time. Low PDE4D mRNA levels correlated negatively with HLA-DMA and HLA-DMB. LPS administration and sepsis are, therefore, associated with different PDE mRNA expression patterns. The effect of PDE changes on immune dysfunction and HLA-DR expression requires further investigation.

Apolipoprotein L Expression Correlates with Neutrophil Cell Death in Critically Ill Patients.

Delayed neutrophil apoptosis has been demonstrated in sepsis and may contribute to organ damage. It has recently been proposed that apolipoprotein L (ApoL) may be involved in programmed cell death, but the expression and functions of ApoLs in leukocytes (especially neutrophils) during sepsis and other inflammatory conditions are currently unknown. In this prospective observational study in a 36-bed university hospital medicosurgical intensive care unit (ICU), we included 78 adult ICU patients with (n = 41) or without (n = 37) sepsis and 47 healthy volunteers. We analyzed ApoL mRNA expression using quantitative polymerase chain reaction in whole blood leukocytes and protein expression in CD15 isolated neutrophils using Western blotting. Neutrophil apoptosis was assessed using the APO-BRDU method. Apolipoprotein L mRNA was downregulated in whole blood leukocytes and neutrophils in ICU patients compared with in healthy volunteers, and this effect translated at the protein level as indicated by Western blot analysis of neutrophils. There was a negative correlation between ApoL expression in neutrophils and C-reactive protein levels and a positive correlation between the number of apoptotic neutrophils and mRNA levels of ApoL1 and ApoL2. The degree of neutrophil apoptosis in critically ill patients is therefore correlated with modified expression profiles of ApoLs.

NFAT-1, Sp-1, Sp-3, and miR-21: New regulators of chemokine C receptor 7 expression in mature human dendritic cells.

The chemokine C receptor 7 (CCR7) is a G-protein-coupled heptahelical receptor (GPCR) that is expressed on a wide variety of cells including memory T cells, B cells, mature dendritic cells, and cancer cells. Activated by its ligands CCL19 or CCL21, CCR7 plays a major role in metastasis of cancer cells. Recent studies demonstrated the role of NF-κB and AP-1 transcription factors in addition to let-7 microRNA in CCR7 expression. Our ChIP assays further show the binding of Sp-1, Sp-3 and NFAT-1 transcription factors to their potential binding sites in the 1Kb promoter region with the later found to inhibit whilst Sp-1, and Sp-3 were found to stimulate CCR7 expression as demonstrated by transfection assays. On the other hand, in addition to the known let-7 regulation of CCR7, we found miR-21 to have a highly conserved target region in CCR7 3'UTR and to be significantly down-regulated during the course of dendritic cell maturation, allowing for high expression of CCR7.