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.

FIG. 1

Chemotactic activities of nonmastitic and mastitic mammary secretions. The data are from a representative experiment with at least three separate nonmastitic or mastitic mammary secretions. Samples were run at physiological concentrations. Data are mean CI values (number of neutrophils which migrated towards sample/number of neutrophils which migrated towards a control medium) ± standard errors of the means. ∗, P < 0.05 compared to the control medium (media).

FIG. 2

Checkerboard analysis of nonmastitic (A) and mastitic (B) mammary secretions. Various concentration gradients were established by placing different dilutions (Neat, undiluted) of samples in the bottom and top (along with responder cells) wells of a Boyden chamber. The data indicate that migration is dependent on an increasing concentration gradient. Data are expressed as the means of triplicate samples. Media, medium alone (used as a negative control).

FIG. 3

Effects of anti-IL-8 antibodies on neutrophil chemotaxis towards nonmastitic and mastitic mammary secretions. Data are representative of at least three separate samples and are expressed as means ± standard errors of the means. ∗, P < 0.05 compared to the control medium (media); ∗∗, P < 0.05 compared to mastitic secretions.

FIG. 4

Chemotactic activities of mastitic milk molecular mass fractions. Data are expressed as means of triplicate samples ± standard errors of the means. ∗, P < 0.05 compared to the control medium (media); ∗∗, P < 0.05 compared to the <10-kDa fraction. The <10-kDa fraction was used at a concentration of 0.1 mg/ml (1 mg/ml was inhibitory to chemotaxis). The >10-kDa fraction was used at a 1-mg/ml concentration, and further dilution had no effect on chemotaxis.