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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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Induction of C5a dependent apoptosis in adrenal medullae after CLP.(A) Analysis of adrenal medulla in sham operated animals (neg ctrl) by the TUNEL technique. (B) Adrenal medullae of sham operated animals preincubated with DNAse before TUNEL staining served as positive control. (C, D) Examination of adrenal medullae obtained 24 hrs after CLP by TUNEL assay. (E, F) Adrenal medullae from animals with dual-blockade of C5aR and C5L2 acquired 24 hrs after CLP. Slides are representative of n≥3 animals per condition.
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pone-0002560-g001: Induction of C5a dependent apoptosis in adrenal medullae after CLP.(A) Analysis of adrenal medulla in sham operated animals (neg ctrl) by the TUNEL technique. (B) Adrenal medullae of sham operated animals preincubated with DNAse before TUNEL staining served as positive control. (C, D) Examination of adrenal medullae obtained 24 hrs after CLP by TUNEL assay. (E, F) Adrenal medullae from animals with dual-blockade of C5aR and C5L2 acquired 24 hrs after CLP. Slides are representative of n≥3 animals per condition.

Mentions: Animals were randomly assigned to 3 different groups: sham animals received abdominal midline incision and manipulation of the cecum (no ligation or puncture) and no treatment. Positive control rats received preimmune rabbit serum (5 ml i.p. 12 hrs before CLP) and were subsequently exposed to CLP. The third group of animals received anti-sera to C5aR and C5L2 12 hrs prior to CLP operation (1∶1 ratio, total of 5 ml, i.p.) followed by CLP. Twenty-four hrs after initiation of CLP, animals were euthanized, adrenal glands surgically removed and immediately embedded and frozen in OCT compound. Frozen sections (4 µm) were prepared from the embedded tissue and subjected to TUNEL analysis. As shown in Figure 1A, sham operated animals did not display adrenomedullary apoptosis and served as negative controls. Histological slides obtained from sham operated rats were preincubated with recombinant DNAse (10 min). The induced DNA strand breaks prior to TUNEL labeling served as positive controls (Figure 1B). When adrenal glands of preimmune rabbit serum treated rats were removed 24 hrs after CLP-induced sepsis and evaluated by TUNEL technique, there was significant apoptosis (Figure 1C, D). In sharp contrast, when both receptors for C5a were blocked by anti-sera to C5aR and C5L2, cells of the adrenal medulla from CLP rats failed to show significant signs of apoptosis (Figure 1E, 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)

Induction of C5a dependent apoptosis in adrenal medullae after CLP.(A) Analysis of adrenal medulla in sham operated animals (neg ctrl) by the TUNEL technique. (B) Adrenal medullae of sham operated animals preincubated with DNAse before TUNEL staining served as positive control. (C, D) Examination of adrenal medullae obtained 24 hrs after CLP by TUNEL assay. (E, F) Adrenal medullae from animals with dual-blockade of C5aR and C5L2 acquired 24 hrs after CLP. Slides are representative of n≥3 animals per condition.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002560-g001: Induction of C5a dependent apoptosis in adrenal medullae after CLP.(A) Analysis of adrenal medulla in sham operated animals (neg ctrl) by the TUNEL technique. (B) Adrenal medullae of sham operated animals preincubated with DNAse before TUNEL staining served as positive control. (C, D) Examination of adrenal medullae obtained 24 hrs after CLP by TUNEL assay. (E, F) Adrenal medullae from animals with dual-blockade of C5aR and C5L2 acquired 24 hrs after CLP. Slides are representative of n≥3 animals per condition.
Mentions: Animals were randomly assigned to 3 different groups: sham animals received abdominal midline incision and manipulation of the cecum (no ligation or puncture) and no treatment. Positive control rats received preimmune rabbit serum (5 ml i.p. 12 hrs before CLP) and were subsequently exposed to CLP. The third group of animals received anti-sera to C5aR and C5L2 12 hrs prior to CLP operation (1∶1 ratio, total of 5 ml, i.p.) followed by CLP. Twenty-four hrs after initiation of CLP, animals were euthanized, adrenal glands surgically removed and immediately embedded and frozen in OCT compound. Frozen sections (4 µm) were prepared from the embedded tissue and subjected to TUNEL analysis. As shown in Figure 1A, sham operated animals did not display adrenomedullary apoptosis and served as negative controls. Histological slides obtained from sham operated rats were preincubated with recombinant DNAse (10 min). The induced DNA strand breaks prior to TUNEL labeling served as positive controls (Figure 1B). When adrenal glands of preimmune rabbit serum treated rats were removed 24 hrs after CLP-induced sepsis and evaluated by TUNEL technique, there was significant apoptosis (Figure 1C, D). In sharp contrast, when both receptors for C5a were blocked by anti-sera to C5aR and C5L2, cells of the adrenal medulla from CLP rats failed to show significant signs of apoptosis (Figure 1E, 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