Date of Award
Doctor of Philosophy
Gina M. Pighetti
Stephen P. Oliver, Tim Sparer, Arnold Saxton, Alan G. Mathew
Mastitis continues to be the most economically devastating disease affecting dairy cows worldwide. Neutrophil recruitment to the mammary gland and neutrophil functional ability once there often determine whether a bacterial infection is eliminated or becomes chronic. A slow or ineffective neutrophil attack allows bacteria to continue their assault and generate a longer lasting and potentially more damaging infection. Since neutrophils are key players in the resolution of mastitis, and a polymorphism in CXCR1 +777 (G→C) has been associated with susceptibility to mastitis, the hypothesis of this research was that different CXCR1 +777 genotypes were associated with efficiency of in vitro neutrophil activity and/or IL-8 receptor expression and intracellular signaling. Neutrophil function (migration, adhesion molecule upregulation, survival from spontaneous apoptosis, glutathione levels, reactive oxygen generation and bactericidal activity) as well as CXCR1 and CXCR2 expression and signaling were evaluated in cows with different CXCR1 +777 genotypes. Cows with a CC genotype, which have previously shown increased susceptibility to mastitis, had decreased adhesion molecule upregulation and neutrophil migration towards IL-8 compared to cows with a GG genotype. Furthermore, cows with a CC genotype displayed decreased ROS generation and, paradoxically, increased survival from spontaneous apoptosis. In addition, initial observations revealed that overall IL-8 receptor numbers tended to be lower in cows with a CC genotype compared to cows with a GG genotype. However, in the presence of a CXCR2 inhibitor (SB225002), CXCR1 affinity was about 5-fold lower in cows with a CC genotype and this may have resulted in an underestimation of receptor numbers in cows with this genotype. Additionally, intracellular calcium ([Ca++]i) release was lower in cows with a CC genotype when cells were stimulated with IL-8 but not epithelial derived neutrophil attractant-78. When neutrophils were stimulated with an optimal dose of IL-8 in the presence of SB225002, allowing only signaling through CXCR1, [Ca++]i release was lower in cows with a CC genotype, suggesting decreased CXCR1 signaling in these animals, potentially due to lower CXCR1 affinity for IL-8. This research provides evidence for neutrophil functional differences and differential CXC receptor activity and signaling in cows with specific CXCR1 genotypes. Future research aimed at better characterizing CXCR1 and CXCR2 in the bovine as well as corroboration of these findings in an in vivo model may provide prospective ways to enhance or regulate neutrophil function in dairy cows and potentially increase their resistance to mastitis and other inflammatory diseases.
Rambeaud, Magdalena, "Differential Neutrophil Function and Intracellular Signaling in Dairy Cows with Specific CXCR1 Genotypes. " PhD diss., University of Tennessee, 2006.