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The complement anaphylatoxin C5a induces apoptosis in adrenomedullary cells during experimental sepsis.

Flierl MA, Rittirsch D, Chen AJ, Nadeau BA, Day DE, Sarma JV, Huber-Lang MS, Ward PA - PLoS ONE (2008)

Bottom Line: These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2.PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner.Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
Sepsis remains a poorly understood, enigmatic disease. One of the cascades crucially involved in its pathogenesis is the complement system. Especially the anaphylatoxin C5a has been shown to have numerous harmful effects during sepsis. We have investigated the impact of high levels of C5a on the adrenal medulla following cecal ligation and puncture (CLP)-induced sepsis in rats as well as the role of C5a on catecholamine production from pheochromocytoma-derived PC12 cells. There was significant apoptosis of adrenal medulla cells in rats 24 hrs after CLP, as assessed by the TUNEL technique. These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2. When rats were subjected to CLP, levels of C5a and norepinephrine were found to be antipodal as a function of time. PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner. This impaired production could be related to C5a-induced initiation of apoptosis as defined by binding of Annexin V and Propidium Iodine to PC12 cells. Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro. These data suggest that experimental sepsis induces apoptosis of adrenomedullary cells, which are responsible for the bulk of endogenous catecholamines. Septic shock may be linked to these events. Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis.

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C5a-dependent induction of apoptosis in PC12 cells.(A, B) Incubation of PC12 cells in growth medium (30 min, 37°C) with subsequent analysis for apoptotic events by Propidium Iodine and Annexin V. (C, D) Preincubation of PC12 cells with the inactive derivate of the pan-caspase inhibitor ZVAD (1 hr, 37°C), followed by medium change, exposure to 10 nM rrC5a (30 min, 37°C) and staining for Propidium Iodine and Annexin V. (E, F) PC12 cells were pretreated with the active derivate of the pan-caspase inhibitor ZVAD (1 hr, 37°C), with subsequent medium change and incubation with 10 nM rrC5a (30 min, 37°C) and staining with Propidium Iodine and Annexin V. Staining was assessed using fluorescing microscopy and digital imaging. Slides are representative of n = 3 per condition.
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pone-0002560-g004: C5a-dependent induction of apoptosis in PC12 cells.(A, B) Incubation of PC12 cells in growth medium (30 min, 37°C) with subsequent analysis for apoptotic events by Propidium Iodine and Annexin V. (C, D) Preincubation of PC12 cells with the inactive derivate of the pan-caspase inhibitor ZVAD (1 hr, 37°C), followed by medium change, exposure to 10 nM rrC5a (30 min, 37°C) and staining for Propidium Iodine and Annexin V. (E, F) PC12 cells were pretreated with the active derivate of the pan-caspase inhibitor ZVAD (1 hr, 37°C), with subsequent medium change and incubation with 10 nM rrC5a (30 min, 37°C) and staining with Propidium Iodine and Annexin V. Staining was assessed using fluorescing microscopy and digital imaging. Slides are representative of n = 3 per condition.

Mentions: To evaluate whether paralysis of catecholamine release from PC12 cells might be related to induction of apoptosis by rrC5a, PC12 cells were separated into three experimental groups: the negative control group was pretreated (1 hr, 37°C) and incubated (30 min, 37°C) in growth medium only. A positive control group received pretreatment (1 hr, 37°C) with the inactive derivative of the pan-caspase inhibitor ZVAD [Z-VAD-FMK(non-omethylated)] followed by medium change and subsequent incubation with 10 nM rrC5a (30 min, 37°C). A third group was pretreated for 1 hr at 37°C with the active derivative of the pan-caspase inhibitor ZVAD [Z-VAD(Ome)-FMK] followed by consecutive medium change and exposure 10 nM rrC5a (30 min, 37°C). Following incubation, PC12 cells were stained with Propidium Iodine or Annexin V. As depicted in Figure 4, negative control cells displayed virtually no staining for either Propidium Iodine or Annexin V (panels A, B). When cells were pretreated with inactive ZVAD and subsequently exposed to rrC5a (10 nM), there was a substantial fluorescence with Propidium Iodine or Annexin V stains (Figure 4C, D), indicating induction of apoptosis by rrC5a. These C5a-dependent effects were significantly reduced when PC12 cells were pretreated with the active derivate of the pan-caspase inhibitor, ZVAD (Figure 4E, F).


The complement anaphylatoxin C5a induces apoptosis in adrenomedullary cells during experimental sepsis.

Flierl MA, Rittirsch D, Chen AJ, Nadeau BA, Day DE, Sarma JV, Huber-Lang MS, Ward PA - PLoS ONE (2008)

C5a-dependent induction of apoptosis in PC12 cells.(A, B) Incubation of PC12 cells in growth medium (30 min, 37°C) with subsequent analysis for apoptotic events by Propidium Iodine and Annexin V. (C, D) Preincubation of PC12 cells with the inactive derivate of the pan-caspase inhibitor ZVAD (1 hr, 37°C), followed by medium change, exposure to 10 nM rrC5a (30 min, 37°C) and staining for Propidium Iodine and Annexin V. (E, F) PC12 cells were pretreated with the active derivate of the pan-caspase inhibitor ZVAD (1 hr, 37°C), with subsequent medium change and incubation with 10 nM rrC5a (30 min, 37°C) and staining with Propidium Iodine and Annexin V. Staining was assessed using fluorescing microscopy and digital imaging. Slides are representative of n = 3 per condition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2481299&req=5

pone-0002560-g004: C5a-dependent induction of apoptosis in PC12 cells.(A, B) Incubation of PC12 cells in growth medium (30 min, 37°C) with subsequent analysis for apoptotic events by Propidium Iodine and Annexin V. (C, D) Preincubation of PC12 cells with the inactive derivate of the pan-caspase inhibitor ZVAD (1 hr, 37°C), followed by medium change, exposure to 10 nM rrC5a (30 min, 37°C) and staining for Propidium Iodine and Annexin V. (E, F) PC12 cells were pretreated with the active derivate of the pan-caspase inhibitor ZVAD (1 hr, 37°C), with subsequent medium change and incubation with 10 nM rrC5a (30 min, 37°C) and staining with Propidium Iodine and Annexin V. Staining was assessed using fluorescing microscopy and digital imaging. Slides are representative of n = 3 per condition.
Mentions: To evaluate whether paralysis of catecholamine release from PC12 cells might be related to induction of apoptosis by rrC5a, PC12 cells were separated into three experimental groups: the negative control group was pretreated (1 hr, 37°C) and incubated (30 min, 37°C) in growth medium only. A positive control group received pretreatment (1 hr, 37°C) with the inactive derivative of the pan-caspase inhibitor ZVAD [Z-VAD-FMK(non-omethylated)] followed by medium change and subsequent incubation with 10 nM rrC5a (30 min, 37°C). A third group was pretreated for 1 hr at 37°C with the active derivative of the pan-caspase inhibitor ZVAD [Z-VAD(Ome)-FMK] followed by consecutive medium change and exposure 10 nM rrC5a (30 min, 37°C). Following incubation, PC12 cells were stained with Propidium Iodine or Annexin V. As depicted in Figure 4, negative control cells displayed virtually no staining for either Propidium Iodine or Annexin V (panels A, B). When cells were pretreated with inactive ZVAD and subsequently exposed to rrC5a (10 nM), there was a substantial fluorescence with Propidium Iodine or Annexin V stains (Figure 4C, D), indicating induction of apoptosis by rrC5a. These C5a-dependent effects were significantly reduced when PC12 cells were pretreated with the active derivate of the pan-caspase inhibitor, ZVAD (Figure 4E, F).

Bottom Line: These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2.PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner.Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
Sepsis remains a poorly understood, enigmatic disease. One of the cascades crucially involved in its pathogenesis is the complement system. Especially the anaphylatoxin C5a has been shown to have numerous harmful effects during sepsis. We have investigated the impact of high levels of C5a on the adrenal medulla following cecal ligation and puncture (CLP)-induced sepsis in rats as well as the role of C5a on catecholamine production from pheochromocytoma-derived PC12 cells. There was significant apoptosis of adrenal medulla cells in rats 24 hrs after CLP, as assessed by the TUNEL technique. These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2. When rats were subjected to CLP, levels of C5a and norepinephrine were found to be antipodal as a function of time. PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner. This impaired production could be related to C5a-induced initiation of apoptosis as defined by binding of Annexin V and Propidium Iodine to PC12 cells. Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro. These data suggest that experimental sepsis induces apoptosis of adrenomedullary cells, which are responsible for the bulk of endogenous catecholamines. Septic shock may be linked to these events. Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis.

Show MeSH
Related in: MedlinePlus