Monocyte apoptosis in experimental sepsis by multidrug-resistant species

Abstract number: 1134_04_133

Antonopoulou A., Poukoulidou T., Adamis T., Tziortzioti V., Tsaganos T., Giamarellou H., Giamarellos-Bourboulis E.J.

Objectives:  

There is little evidence regarding monocyte apoptosis during the course of sepsis. This study aims to investigate changes in cytokine release by peripheral blood monocytes and their correlation to monocyte apoptosis.

Methods:  

Acute pyelonephritis was induced in 20 New Zealand rabbits after ligation of the right pyelo-ureteral junction; all were inoculated of an 8log10 cfu/ml inoculum in the renal pelvis, 10 of a multidrug-resistant clinical isolate of Pseudomonasaeruginosa and 10 of a multidrug-resistant isolate of Klebsiellapneumonia. Blood was sampled for estimation of tumor necrosis factor-alpha (TNFa) and for isolation of monocytes. Mononuclear cells were isolated after centrifugation over Ficoll and incubated for one hour at 37°C under 5% CO2 in RPMI with 10% FBS and 2 mM glutamine. Non-adherent cells were removed and after trypsinization monocytes were lyzed. TNF-alpha was estimated by a bioassay on L929 fibrosarcoma cell line; caspase-3 activity was estimated in the cytosolic extract by a kinetic chromogenic assay.

Results:  

Mean ± SE values for serum TNF-alpha at 4 and 24 hours after challenge by P.aeruginosa were 37.97 ± 28.07, and 16.19 ± 10.62 pg/ml respectively. At the same intervals, mean values for caspase-3 activity were 6472.74 ± 4850.7 and 1129.48 ± 863.35 pmol/min.10000cells respectively. Mean ± SE values for serum TNF-alpha at 4 and 24 hours after challenge by K. pneumoniae were 253.97 ± 170.15, and 83.77 ± 62.30 pg/ml respectively. At the same intervals, mean values for caspase-3 activity were 8.95 ± 4.40 and 10.47 ± 5.87 pmol/min.10000 cells respectively.

Conclusion:  

Experimental acute pyelonephritis by multidrug-resistant Pseudomonasaeruginosa is accompanied by enhanced activity of monocyte caspase-3 over the first hours following bacterial challenge revealing an early initiation of the apoptotic process. Contrary findings are noted for K. pneumoniae. Results merit clinical relevance.