Involvement of TNF-α and MAPK pathway in the intramammary MMP-9 release via degranulation of cow neutrophils during acute mammary gland involution

Vet Immunol Immunopathol. 2012 Jun 30;147(3-4):161-9. doi: 10.1016/j.vetimm.2012.04.011. Epub 2012 Apr 19.

Abstract

Neutrophil-derived MMP-9 activity is regulated more promptly and efficiently at the level of degranulation than at other levels of regulation. In human neutrophils, degranulation is one of the earliest responses to TNF-α stimulation, which involves protein kinase C and mitogen-activating protein kinase (MAPK) pathways. The level of MMP-9 in mammary secretion of cows increases drastically following milk-stasis, which is partially explained by increases of both neutrophil infiltration and neutrophil degranulation per se. Since MMP-9 represents one of the major remodeling capacities in the mammary gland of cows during early dry period, the current study attempted to explore the involved intracellular mechanisms in the up-regulated MMP-9 secretion. We repeatedly measured on the somatic cells of mammary secretion along the early dry period of cows the expression of TNF-α protein and the phosphorylation of p38 MAPK, ERK, and JNK. Also, cultures of bovine peripheral neutrophils were conducted to examine the mode of short-term MMP-9 secretion in response to TNF-α stimulation and the blocking effects of TNF-α antibody and inhibitors of MAPK pathways. Ex vivo measurements show that conventional cow milk has fully transformed into a neutrophil-abundant, lactoferrin-rich, and high-MMP-9 mammary secretion by d 7 in milk-stasis. No significant (P>0.05) change, however, was found in the expression of TNF-α or the phosphorylation extent of MAPK pathway intermediates on the somatic cells of mammary secretion during the first 3 weeks in milk-stasis. In vitro studies indicate linear increase of short-term MMP-9 release in response to TNF-α stimulation in dosages between 0.1 and 10 ng/ml. In the presence of preparations of d 7-dry secretion of cows, the short-term release of MMP-9 from bovine peripheral neutrophils was significantly (P<0.05) blocked by inhibitor of p38 MAPK but was significantly (P<0.05) promoted by ERK inhibitor while TNF-α antibody or JNK inhibitor exerted no effect. In conclusion, the current ex vivo measurements suggest no apparent association of TNF-α and MAPK pathway with long term intramammary accumulation of MMP-9 during the early dry period of cows, whereas cultures of bovine peripheral neutrophils under a simulated acute involution intramammary environment of cows suggest a role played by TNF-α and MAPK pathways in the short-term MMP-9 release via degranulation.

MeSH terms

  • Animals
  • Cattle / anatomy & histology
  • Cattle / immunology*
  • Cattle / physiology*
  • Cell Degranulation / drug effects
  • Cell Degranulation / physiology
  • Female
  • Humans
  • In Vitro Techniques
  • Lactation / immunology
  • Lactation / physiology
  • MAP Kinase Signaling System* / drug effects
  • Mammary Glands, Animal / anatomy & histology
  • Mammary Glands, Animal / immunology
  • Mammary Glands, Animal / physiology*
  • Matrix Metalloproteinase 9 / metabolism*
  • Milk / enzymology
  • Milk / immunology
  • Neutrophils / drug effects
  • Neutrophils / physiology
  • Protein Kinase Inhibitors / pharmacology
  • Tumor Necrosis Factor-alpha / antagonists & inhibitors
  • Tumor Necrosis Factor-alpha / metabolism*
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Protein Kinase Inhibitors
  • Tumor Necrosis Factor-alpha
  • Matrix Metalloproteinase 9