The aim of this study was to examine the production of cytokines during bovine mastitis and their effects on neutrophil function. Cytokine expression (interleukin [IL]-6, IL-8 and IL-10), and neutrophil trafficking and activation were examined in lactating cows 12 hours after intramammary lipopolysaccharide (LPS) infusion (0.2 [mu]g/mL). Interleukin 8 mRNA was detected in the somatic cells (SC) from LPS-infused quarters and from quarters with Gram-positive bacterial mastitis. There was a negative correlation between the SC count and IL-8 expression in LPS induced mastitis, but not in clinical cases. RT-PCR revealed mRNA for IL-8 in SC from both pre- and post-infused quarters, thus suggesting a role for these cells in regulating neutrophil recruitment and activation. Sporadic expression of IL-6, IL-8 and IL-10 was seen in all mammary gland quarters and peripheral tissues examined. Expression of IL-8 mRNA was observed in LPS stimulated monocytes, neutrophils and endothelial cells suggesting these cell types may play a role in IL-8 production during mastitis. The bovine IL-8 gene was cloned and sequenced from LPS stimulated monocyte cultures and is 1411 base pairs long with high predicted protein homology to other species. Human IL-8 protein was used to stimulate bovine neutrophils in vitro. Neutrophils exhibited significant migration in the presence of hrIL-8, and both the 77 and 72 aa forms were equally effective. Migration seen with 20% zymosan activated serum (ZAS) was not significantly different from migration with LPS stimulated monocyte supernatants (CM). Significant degranulation of alkaline phosphatase (ALK-P) (specific granules) was seen, but myeloperoxidase (MPO) release and superoxide anion production were not enhanced in bovine neutrophils by either form of hrIL-8. The ZAS and CM significantly enhanced neutrophil degranulation of ALK-P and MPO as well as superoxide anion production. Circulating peripheral blood neutrophils from LPS infused animals had significant spontaneous and induced degranulation (ALK-P and MPO) and may suggest systemic stimulation of these cells. These studies suggest that bovine IL-8 mRNA is present and may activate neutrophils during bacterial mastitis in lactating cows.