Chemotactic Activities in Nonmastitic and Mastitic Mammary Secretions: Presence of Interleukin-8 in Mastitic but Not Nonmastitic Secretions

Abstract

Due to its association with low-quality milk and a decrease in milk production in bovines, mastitis is a major cause of economic loss. Additionally, mastitis can be harmful to suckling newborns and can cause damage to the mammary gland. In mastitic mammary secretions there is a substantial increase in somatic cells, specifically neutrophils. In this study we examined the ability of mastitic and nonmastitic mammary secretions to cause in vitro neutrophil chemotaxis using a microchemotaxis assay. Also, the role of the inflammatory chemokine interleukin-8 (IL-8) in neutrophil recruitment during mastitis was addressed in these in vitro experiments. We found that both nonmastitic and mastitic mammary secretions were chemotactic, not chemokinetic, for neutrophils. The neutrophil chemotactic activity in mastitic, but not nonmastitic, mammary secretions was blocked by anti-IL-8 antibodies. Molecular mass separation of the active components showed that the chemotactic activity of the mastitic secretions was present in the 10-kDa-or-less fraction and was blocked by anti-IL-8 antibodies. These results indicate that IL-8 plays a major role in neutrophil recruitment during mastitis. An understanding of its role will be of help in designing strategies for immunomodulatory therapies for mastitis.

Increased Nuclear Factor κb Activity in Milk Cells of Mastitis-Affected Cows

“…The expression level of IL-8 and GM-CSF was drastically increased in milk cells of mastitis-affected cows, when compared to that found in milk cells from healthy cows. These findings are consistent with previous data from Barber and Yang (1998), who showed elevated IL-8 concentrations in mastitic secretions. IL-8 is a potent neutrophil chemoattractant (Baggiolini et al, 1989), whereas GM-CSF delays neutrophil apoptosis (Coxon et al, 1999).…”

“…The accumulation and the activation of neutrophils at the site of infection require local expression of many inflammatory proteins, such as: 1) adhesion factors, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which play a cardinal role in neutrophil margination, diapedesis and transepithelial migration; 2) chemokines, such as interleukin (IL)-8, which is potently chemotactic for neutrophils; and 3) cytokines, such as IL-1β and tumor necrosis factor (TNF)-α, which activate neutrophils, and granulocyte/macrophage colony-stimulating factor (GM-CSF), which increases neutrophil survival (Coxon et al, 1999). Increased TNF-α, IL-1β, and IL-8 concentrations 1260 in milk from mastitis-affected cows have been reported, emphasizing the potential role of these proteins in neutrophilic inflammation of the mammary gland (Shuster et al, 1993;Barber and Yang, 1998;Riollet et al, 2001).…”

Inducible and Constitutive In Vitro Neutrophil Chemokine Expression by Mammary Epithelial and Myoepithelial Cells

“…Briefly, 2 ϫ 10 6 blood monocytes were isolated by a Ficoll-diatrizoate gradient (specific density, 1.090) and allowed to adhere onto plastic. Adherent macrophages were stimulated with lipopolysaccharide ( antibodies as described previously (1). Briefly, supernatants were treated with an appropriate dilution of antibodies just prior to being placed in the Boyden chamber.…”

“…In addition, IL-8 has been detected in human mammary secretions, and human maternal cells in breast milk express mRNA for IL-8 (33). IL-8 has also been detected in mammary secretions from glands challenged with Escherichia coli (30,31) and in mastitic mammary secretions (1).…”

“…Mastitic, nonmastitic, and mammary secretions from different lactation stages induce migration of neutrophils (1,16). The migration of neutrophils from the peripheral blood, through the mammary tissue, and into the mammary secretions is called chemotaxis (24).…”