38 research outputs found

    Reduced internalization of TNF-ɑ/TNFR1 down-regulates caspase dependent phagocytosis induced cell death (PICD) in neonatal monocytes

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    <div><p>Phagocytosis-induced cell death (PICD) is diminished in cord blood monocytes (CBMO) as compared to cells from adults (PBMO) due to differences in the CD95-pathway. This may support a prolonged pro-inflammatory response with sequels of sustained inflammation as seen in neonatal sepsis. Here we hypothesized that TNF-α mediated induction of apoptosis is impaired in CBMO due to differences in the TNFR1-dependent internalization. Monocytes were infected with <i>Escherichia coli-</i>GFP (<i>E</i>. <i>coli-</i>GFP). Monocyte phenotype, phagocytic activity, induction of apoptosis, and TNF-α/TNF-receptor (TNFR) -expression were analysed. In the course of infection TNF-α-secretion of CBMO was reduced to 40% as compared to PBMO (p<0.05). Neutralization of TNF-α by an αTNF-α antibody reduced apoptotic PICD in PBMO four-fold (p < 0.05 vs. infection with <i>E</i>. <i>coli</i>). PICD in CBMO was reduced 5-fold compared to PBMO and showed less responsiveness to αTNF-α antibody. CBMO expressed less pro-apoptotic TNFR1, which, after administration of TNF-α or infection with <i>E</i>. <i>coli</i> was internalized to a lesser extent. With similar phagocytic capacity, reduced TNFR1 internalization in CBMO was accompanied by lower activation of caspase-8 (p < 0.05 vs. PBMO). Stronger caspase-8 activation in PBMO caused more activation of effector caspase-3 and apoptosis (all p < 0.05 vs. PBMO). Our results demonstrate that TNFR1 internalization is critical in mediating PICD in monocytes after infection with <i>E</i>.<i>coli</i> and is reduced in CBMO.</p></div

    Internalization of TNFR1 induces caspase-8 and -3 cleavage after <i>E</i>.<i>coli</i> infection.

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    <p>Assessment of TNFR1 internalization in monocytes 2 hrs p.i. (A; n = 11, left panel) and proteolytic cleavage of caspases-8 in monocytes with internalized TNFR1 (A; n = 3,panel with dotted bars;*p < 0.05, ***p < 0.005, clamped bars, student`s t-test; blunt-ended bars, one- and two-way ANOVA). Monocytes which exhibited surface retained TNFR1 (A, panel to the right, hatched bars) were also tested for cleaved caspase-8 (second right panel, both, *p < 0.05, ***p < 0.005, clamped bars, student`s t-test; blunt-ended bars, one- and two-way ANOVA).The percentage of monocytes expressing cleaved caspase-8 is given (B). Pre-treatment with either zVAD or αTNF-α antibody as described (n = 10, *p < 0.05, ***p < 0.005, clamped bars, student`s t-test; blunt-ended bars, two-way ANOVA, brunched clamped bars, one-way ANOVA). The cleavage of RIP was monitored by calculating the quotient of RIP/RIPc after quantification of RIP and RIPc signals. If RIPc outweighs RIP the quotient is less than 1 which is the value found in non-treated probes (C; n = 3; *p < 0.05, **p < 0.01, ***p < 0.005, clamped bars, student`s t-test, blunt-ended bars, two-way ANOVA).</p

    TNFR1 expression after infection is functional in induction of monocytic apoptosis.

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    <p>TNFR1 expression on the plasma-membrane of PBMO and CBMO before and after infection depicted as mean expression of TNFR1 on all monocytes (A, n = 6; *p < 0.05, **p < 0.01, ***p < 0.005, clamped bars, student`s t-test; blunt-ended bars, two-way ANOVA). To groups indicated, the metalloprotease inhibitor GM6001 was added. TNF-α secretion is diminished by GM6001 (B, n = 3; *p < 0.05, **p < 0.01, ***p < 0.005, clamped bars, student`s t-test). Detection of apoptotic PBMO and CBMO after infection and/or addition of GM6001 and TNF-α (C, n = 8, *p < 0.05, **p < 0.01, clamped bars, student`s t-test *within bars, p < 0.005, two-way ANOVA).</p

    Bystander apoptosis occurs without cellular contact to the phagocyting monocyte.

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    <p>Monocytes were infected with <i>E. coli</i>-GFP and GFP –positive and -negative cells were separated by FACS sorting. Apoptosis was assessed 24 hours p.i. by quantification of hypodiploid DNA (Fig. 3A; n = 3, *p<0.05). Monocytes were infected with <i>E.coli</i>-GFP for one hour and free bacteria were removed. CFSE labelled monocytes of the same donor were co-incubated for 4 hours in a concentration of 10∶1. Un-infected, unlabelled monocytes served as controls. Apoptosis was assessed 4 hours p.i. in GFP+ and CFSE+ monocytes (B; n = 5). Monocytes were infected with <i>E. coli</i>-GFP as described (i.e. 1 hour w/o antibiotics and 3–21 hours with gentamycin) in a transwell setup for the time intervals indicated (“<i>cis</i>” chamber, C, sketch to the right); non-infected cells served as controls. Monocytes from the same donor were co-cultivated in compartments, separated by teflon membranes (“<i>trans</i>” chamber). Apoptosis was detected by assessment of hypodiploid DNA-content (C, n = 5; * p<0.05, ** = p<0.001). In the same experimental setup, a CFU-assay was performed by lysis of cells (Fig. 3D, n = 5; * p<0.05, ** p<0.001).</p

    Transport to the phagolysosome is not obligatory for monocyte apoptosis.

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    <p>The gating strategy and nomenclature is shown by dot-plot analysis of monocytes, infected with EOS-FP <i>E.coli</i> for the indicated time intervals (A). Bound bacteria exhibit a red fluorescence, in the FL-2 channel (red bacteria in the micrograph, gated BI). Bacteria, transported to the phagolysosome, exhibit a green fluorescence in the FL-1 channel (green bacteria in the micrograph, gate P). Monocytes without bacterial contact were gated in NC. The chart (B) summarizes the proceeding the phagocytic process within the indicated time intervals. Details are given in the supplement. (C): Assessment of monocyte apoptosis by hypodiploid DNA content in total (second column) and in the three subfractions described (n = 7; ** = p<0.001).</p
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