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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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Impaired production of catecholamines by PC12 cells following C5a exposure.Norepinephrine levels in PC12 cell supernatants incubated with or without recombinant rat C5a (10 nM; rrC5a) as a function of time (A). PC12 cell supernatants incubated with or without 10 nM rrC5a subjected to dopamine analysis (B). Levels of norepinephrine (C) and dopamine (D) in PC12 cell supernatants following 30 min incubation with increasing doses of rrC5a (0 nM–100 nM). (E) Analysis of dopamine levels in PC12 cell supernatants after incubation with either LPS (50 ng/ml) or pepstatin (50 ng/ml) as a function of time. All bars are presented as mean±s.e.m. For each bar n = 5.
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pone-0002560-g003: Impaired production of catecholamines by PC12 cells following C5a exposure.Norepinephrine levels in PC12 cell supernatants incubated with or without recombinant rat C5a (10 nM; rrC5a) as a function of time (A). PC12 cell supernatants incubated with or without 10 nM rrC5a subjected to dopamine analysis (B). Levels of norepinephrine (C) and dopamine (D) in PC12 cell supernatants following 30 min incubation with increasing doses of rrC5a (0 nM–100 nM). (E) Analysis of dopamine levels in PC12 cell supernatants after incubation with either LPS (50 ng/ml) or pepstatin (50 ng/ml) as a function of time. All bars are presented as mean±s.e.m. For each bar n = 5.

Mentions: Rat PC12 cells derive from a catecholamine-producing neuroendocrine tumor of the adrenal medulla, termed a pheochromocytoma. They secrete norepinephrine and dopamine, but not epinephrine [16]. As shown in Figure 3A, cells not otherwise treated released norepinephrine, with levels peaking at 30 min and gradually declining thereafter. In contrast, incubation of PC12 cells with 10 nM recombinant rat C5a (rrC5a) completely abolished norepinephrine release over a long period of time (the entire 12 hr time span). Similar inhibitory effects were observed on dopamine release from pheochromocytoma cells (Figure 3B). The abridged secretion of norepinephrine and dopamine by PC12 cells following rrC5a exposure was dose dependent, reaching the inhibitory peak at a concentration of 10 nM rrC5a (Figure 3C, D). To assess if the presence of any non-specific protein had a similar effect on catecholamine release by PC12 cells, PC12 cells were incubated with pepstatin, a protein of similar size to rrC5a, as previously described [17]. Incubation of PC12 cells with pepstatin failed to significantly alter the time-course of dopamine release from pheochromocytoma PC12 cells, as demonstrated in Figure 3E. Moreover, exposure of PC12 cells to lipopolysaccharide (LPS, 50 ng/ml) led to only a transient decrease of dopamine release, returning to baseline levels after 2 hrs (Figure 3E), indicating that the long-term inhibition of catecholamine release by PC12 cells was unique to C5a. Since PC12 cells do not produce epinephrine [16], we were unable to evaluate its secretion.


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)

Impaired production of catecholamines by PC12 cells following C5a exposure.Norepinephrine levels in PC12 cell supernatants incubated with or without recombinant rat C5a (10 nM; rrC5a) as a function of time (A). PC12 cell supernatants incubated with or without 10 nM rrC5a subjected to dopamine analysis (B). Levels of norepinephrine (C) and dopamine (D) in PC12 cell supernatants following 30 min incubation with increasing doses of rrC5a (0 nM–100 nM). (E) Analysis of dopamine levels in PC12 cell supernatants after incubation with either LPS (50 ng/ml) or pepstatin (50 ng/ml) as a function of time. All bars are presented as mean±s.e.m. For each bar n = 5.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002560-g003: Impaired production of catecholamines by PC12 cells following C5a exposure.Norepinephrine levels in PC12 cell supernatants incubated with or without recombinant rat C5a (10 nM; rrC5a) as a function of time (A). PC12 cell supernatants incubated with or without 10 nM rrC5a subjected to dopamine analysis (B). Levels of norepinephrine (C) and dopamine (D) in PC12 cell supernatants following 30 min incubation with increasing doses of rrC5a (0 nM–100 nM). (E) Analysis of dopamine levels in PC12 cell supernatants after incubation with either LPS (50 ng/ml) or pepstatin (50 ng/ml) as a function of time. All bars are presented as mean±s.e.m. For each bar n = 5.
Mentions: Rat PC12 cells derive from a catecholamine-producing neuroendocrine tumor of the adrenal medulla, termed a pheochromocytoma. They secrete norepinephrine and dopamine, but not epinephrine [16]. As shown in Figure 3A, cells not otherwise treated released norepinephrine, with levels peaking at 30 min and gradually declining thereafter. In contrast, incubation of PC12 cells with 10 nM recombinant rat C5a (rrC5a) completely abolished norepinephrine release over a long period of time (the entire 12 hr time span). Similar inhibitory effects were observed on dopamine release from pheochromocytoma cells (Figure 3B). The abridged secretion of norepinephrine and dopamine by PC12 cells following rrC5a exposure was dose dependent, reaching the inhibitory peak at a concentration of 10 nM rrC5a (Figure 3C, D). To assess if the presence of any non-specific protein had a similar effect on catecholamine release by PC12 cells, PC12 cells were incubated with pepstatin, a protein of similar size to rrC5a, as previously described [17]. Incubation of PC12 cells with pepstatin failed to significantly alter the time-course of dopamine release from pheochromocytoma PC12 cells, as demonstrated in Figure 3E. Moreover, exposure of PC12 cells to lipopolysaccharide (LPS, 50 ng/ml) led to only a transient decrease of dopamine release, returning to baseline levels after 2 hrs (Figure 3E), indicating that the long-term inhibition of catecholamine release by PC12 cells was unique to C5a. Since PC12 cells do not produce epinephrine [16], we were unable to evaluate its secretion.

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