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Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils

Mieke Stevens (UGent) , Mario Van Poucke (UGent) , Luc Peelman (UGent) , P Rainard, Bart De Spiegeleer (UGent) , Carolien Rogiers (UGent) , Gerlinde Van de Walle (UGent) , Luc Duchateau (UGent) and Christian Burvenich (UGent)
(2011) JOURNAL OF DAIRY SCIENCE. 94(1). p.152-164
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Abstract
It is well known that signaling in neutrophils through both the complement component 5a (C5a) and C5a receptor (C5aR) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C5a. The aim of the current study was to elucidate the effect of C5a on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C5a, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C5a resulted in a biphasic C5aR expression profile, a phenomenon that might be explained by internalization (at 10 min PI) with subsequent reconstitution (starting at 40 min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C5aR. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKK alpha, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C5aR mRNA at 80 min PI was accompanied by a peak in IL8 mRNA, indicating that C5a is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-kappa B mediated transcription, implying that C5a regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C5aR and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C5aR at 80 min PI, when C5aR and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C5a is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between complement activation and TLR4 signaling were found in the present study.
Keywords
HUMAN POLYMORPHONUCLEAR LEUKOCYTES, NECROSIS-FACTOR-ALPHA, NF-KAPPA-B, GENE-EXPRESSION, C5A RECEPTOR, COMPLEMENT RECEPTOR-3, ESCHERICHIA-COLI, TYROSINE KINASE, UP-REGULATION, KEY ROLE, complement component 5a, neutrophil, sepsis, toll-like receptor 4

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Chicago
Stevens, Mieke, Mario Van Poucke, Luc Peelman, P Rainard, Bart De Spiegeleer, Carolien Rogiers, Gerlinde Van de Walle, Luc Duchateau, and Christian Burvenich. 2011. “Anaphylatoxin C5a-induced Toll-like Receptor 4 Signaling in Bovine Neutrophils.” Journal of Dairy Science 94 (1): 152–164.
APA
Stevens, Mieke, Van Poucke, M., Peelman, L., Rainard, P., De Spiegeleer, B., Rogiers, C., Van de Walle, G., et al. (2011). Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils. JOURNAL OF DAIRY SCIENCE, 94(1), 152–164.
Vancouver
1.
Stevens M, Van Poucke M, Peelman L, Rainard P, De Spiegeleer B, Rogiers C, et al. Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils. JOURNAL OF DAIRY SCIENCE. 2011;94(1):152–64.
MLA
Stevens, Mieke, Mario Van Poucke, Luc Peelman, et al. “Anaphylatoxin C5a-induced Toll-like Receptor 4 Signaling in Bovine Neutrophils.” JOURNAL OF DAIRY SCIENCE 94.1 (2011): 152–164. Print.
@article{1235094,
  abstract     = {It is well known that signaling in neutrophils through both the complement component 5a (C5a) and C5a receptor (C5aR) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C5a. The aim of the current study was to elucidate the effect of C5a on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C5a, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C5a resulted in a biphasic C5aR expression profile, a phenomenon that might be explained by internalization (at 10 min PI) with subsequent reconstitution (starting at 40 min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C5aR. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKK alpha, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C5aR mRNA at 80 min PI was accompanied by a peak in IL8 mRNA, indicating that C5a is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-kappa B mediated transcription, implying that C5a regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C5aR and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C5aR at 80 min PI, when C5aR and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C5a is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between complement activation and TLR4 signaling were found in the present study.},
  author       = {Stevens, Mieke and Van Poucke, Mario and Peelman, Luc and Rainard, P and De Spiegeleer, Bart and Rogiers, Carolien and Van de Walle, Gerlinde and Duchateau, Luc and Burvenich, Christian},
  issn         = {0022-0302},
  journal      = {JOURNAL OF DAIRY SCIENCE},
  keyword      = {HUMAN POLYMORPHONUCLEAR LEUKOCYTES,NECROSIS-FACTOR-ALPHA,NF-KAPPA-B,GENE-EXPRESSION,C5A RECEPTOR,COMPLEMENT RECEPTOR-3,ESCHERICHIA-COLI,TYROSINE KINASE,UP-REGULATION,KEY ROLE,complement component 5a,neutrophil,sepsis,toll-like receptor 4},
  language     = {eng},
  number       = {1},
  pages        = {152--164},
  title        = {Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils},
  url          = {http://dx.doi.org/10.3168/jds.2010-3358},
  volume       = {94},
  year         = {2011},
}

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