TRAIL-treatment improved survival of CASP.

<p>This effect was abrogated by depleting neutrophils. A: Survival of CASP is depicted as Kaplan Meier curves. Mice were treated with anti-Ly6G 24 hrs before CASP induction (anti-Ly6G) to deplete neutrophils. Controls received appropriate isotype controls (isotype). Neutrophil-depleted (anti-Ly6G, TRAIL) and untreated mice (TRAIL) received TRAIL (1 µg/g (wt/wt)) 1 h, 24 h and 48 h after CASP intravenously. TRAIL treatment significantly improved survival of sepsis in previously untreated mice (p<0.001). However, TRAIL-treatment was ineffective in Ly6G-depleted mice. B: Depletion of neutrophils was confirmed via FACS analyses. Representative data 48 hrs after neutrophil depletion are shown. The oval indicates neutrophils detected via CD11b+Ly6Cmed expression.</p

TRAIL-treatment led to induction of apoptosis in neutrophils in sepsis.

<p>A) Spleens, livers and lungs of septic saline-treated (CASP+saline) and septic TRAIL-treated (CASP+TRAIL) were analyzed 20 h after induction of CASP. Sections were stained for Ly6G. Ly6G-positive cells of respective organs (n = 5/group for each organ) were counted in three HPFs and the mean was calculated. The number of neutrophils per HPF is depicted. Box plots and outliers are shown. The infiltration of neutrophils within the septic organs is significantly decreased by TRAIL-treatment in sepsis. Results are representative of two independent experiments. B) The number of apoptotic cells within the spleen, liver and lungs was determined by immunohistochemistry (n = 5/group for each organ, mean of 3 HPFs). TUNEL-straining was performed 20 hours after CASP. Box plots and outliers are shown. TRAIL-treatment decreased the number of apoptotic cells. Results are representative of two experiments performed independently. C) Apoptotic neutrophils were detected by staining Ly6G and TUNEL. The number of apoptotic neutrophils within the respective septic organs 20 hrs after induction of CASP was counted in three HPFs and the mean was calculated (n = 5/group for each organ). Additionally, the number of total apoptotic cells per HPF was counted. The ratio of apoptotic neutrophils over all apoptotic cells was calculated for each HPF. Box plots and outliers are depicted. TRAIL-treatment increased the fraction of apoptotic neutrophils 20 hrs after induction of CASP within the septic organs. D) Representative immunohistochemical analysis of Ly6G (green) and TUNEL (red) in spleens of septic mice 20 hrs after induction of CASP with (right) and without (left) TRAIL-treatment. Apoptotic neutrophils appear yellow. *p<0.05.</p

TRAIL-treatment did not influence cell viability <i>in vitro</i>.

<p>LPS-stimulation increased TRAIL-expression by splenocytes. A: Cultures of splenocytes were stimulated with TRAIL (100 ng/ml) for 48 hours. Cell viability was determined using a CellTiter Blue Assay. Box plots and outliers are depicted. TRAIL-stimulation did not alter the viability of splenocytes. n = 5/group; results are representative of two independently performed experiments. B: Cultures of splenocytes were stimulated with LPS (1 µg/ml) for 24 hours. TRAIL-expression was determined by FACS analyses. Isotype controls were used for background staining. Box plots and representative histograms of FACS analyses are shown. LPS stimulation significantly increased the expression of TRAIL on the cell surface of splenocytes. One of two experiments in which similar results were obtained is shown. *: p<0.05.</p

CASP led to increased expression of DR5 and TRAIL.

<p>DR5 was predominantly expressed on neutrophil cell surface. A: The fraction of neutrophils within the spleen was determined by FACS analyses in non-treated mice (no treatment) versus septic mice 20 hours after induction of CASP (CASP). Box plots are shown. n = 5/group. Results are representative of three independent experiments. B: DR5-expression as well as Ly6G-expression by murine splenocytes was determined by FACS analysis (n = 5). The expression of DR5 on the cell surface of Ly6G+ -splenocytes was compared to the expression of DR5 on all splenocytes. Boxplots are shown. Isotype controls were used for background staining. One of two experiments in which similar results were obtained is shown. C: Representative FACS density plots of the expression of DR5 by splenocytes are shown. Plots were gated on B220 and Ly6G respectively. D: TRAIL-binding on the cell surface of splenocytes is shown as determined by FACS analysis of spleens of untreated and septic mice (CASP; 20 h after CASP) (n = 5/group). Isotype controls were used for background staining. Box plots and outliers are depicted. TRAIL-expression was significantly increased during CASP. Results are representative of three independent experiments. E: TRAIL was stained in spleens of septic TRAIL-treated mice via immunohistochemistry. TRAIL was mainly detected in cells of the splenic red pulp (brown coloured cells, n = 5). One representative picture of five is depicted. A 200x magnification is shown. *p<0.05.</p