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item SOHN, EUN
item Paape, Max
item Bannerman, Douglas
item Connor, Erin
item Fetterer, Raymond

Submitted to: Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/3/2006
Publication Date: 1/1/2007
Citation: Sohn, E.J., Paape, M.J., Bannerman, D.D., Connor, E.E., Fetterer, R.H., Peters, R.R. 2007. Shedding of sCD14 by bovine neutrophils following activation with bacterial lipopolysaccharide results in down-regulation of IL-8. Vet. Res. 38(1):95-108.

Interpretive Summary: Bacteria that pass through the teat end and enter the teat cistern meet the second line of defense: phagocytic leukocytes. Phagocytes, consisting of neutrophils and macrophages, ingest and kill mastitis pathogens. Bacterial invasion and growth within the mammary gland is the main cause of mastitis. Invading bacteria settle next to the epithelial cells lining the mammary ducts, absorbing nutrients from milk while expelling harmful toxins that attack and destroy the epithelium. While helpless against the invading horde of bacteria, the epithelium and white blood cells called macrophages soon release chemical messengers called cytokines that signal the body for help. Soon neutrophils, a specialized form of white blood cells, migrate directly from blood into the bacterial hoard. The PMN release potent oxidants that destroy not only some of the bacteria but also some of the epithelial cells lining the ducts and alveoli within the udder, which will result in lost milk production. Scientists at the USDA, Beltsville discovered that neutrophils also release a protein called CD14, and that this protein suppresses release of the cytokine IL-8, a potent recruiter of neutrophils. This results in reduced migration of neutrophils into the udder and protects the udder from excessive tissue damage.

Technical Abstract: CD14, the leukocyte receptor for lipopolysaccharide (LPS), is important in the response of PMN to infection by Gram-negative bacteria. IL-8 is a potent chemoattractant of PMN. The objective of the present study was to characterize bovine PMN cell surface expression and shedding of CD14 molecules, and its effect on secretion of IL-8 by PMN. PMN were isolated from blood collected from sixteen lactating cows. PMN (5x106 /mL) were cultured in RPMI and stimulated with 0, 1, 10 and 100 ug/mL of LPS for 20 h at 37 degrees C. The percentage of PMN expressing membrane (m) CD14 was determined by flow cytometry. Concentrations of CD14 and IL-8 in RPMI were quantitated by ELISA. To determine the effect of cell density of PMN on release of CD14 and IL-8, varying concentrations of PMN were stimulated with either 0.1 or 10 ug/mL of LPS, and supernatants assayed for CD14 and IL-8. Expression of mCD14 decreased from 35% (control PMN) to 25% after exposure to 100 ug/mL of LPS. Secretion of CD14 increased with increasing cell density and with increasing concentrations of LPS, whereas secretion of IL-8 decreased. Results from quantitative real-time PCR indicated that CD14 gene expression did not increase after stimulation of PMN with LPS. Results from this study indicate that release of CD14 suppressed secretion of IL-8, and that the increase in CD14 resulted from either the shedding of mCD14 or originated from an intracellular pool of sCD14. The suppression of IL-8 by CD14 may be an important mechanism to control influx of PMN into the bovine mammary gland.