Kinetics of cells and cytokines during immune-mediated inflammation in the mammary gland of cows systemically immunized with Staphylococcus aureus alpha-toxin.

Abstract

To examine changes in inflammatory mediators, lymphocyte subpopulations and neutrophil activation that occur during an immune-mediated recruitment of neutrophils in the mammary gland. 11 clinically healthy cows. 5 cows received 2 subcutaneous injections of 30 microg of alpha-toxin of Staphylococcus aureus, two months apart. Three months after the last immunization, 5 microg of alpha-toxin were injected, via the teat end, in one randomly selected quarter of the 5 immunized cows and of the 6 unimmunized cows (control group). Blood and milk samples were collected at several times during 4 days post-challenge. Blood and milk cells were purified to be stained with specific mAbs and analysed by flow cytometry, or to be used for cytokine RT-PCR. Bovine serum albumin, haptoglobin, cytokines and C5a were also analysed in milk or plasma samples using radial immunodiffusion assay or ELISA. Large amounts of cells (> 1 million/ml of milk) were recruited in the quarters of the immunized cows, whereas no recruitment occurred in the control group. In blood of immunized animals, haptoglobin was present and expression of surface adhesion molecules on neutrophils was modified whereas no change was observed concerning the lymphocyte subpopulations. On milk-derived neutrophils, the expression of CD11b and CD18 was upregulated compared to blood, in contrast to CD62L that was downregulated. The CD8+ cells were recruited as soon as 12 h post-challenge, in contrast to the CD4+ cells, 96 h post-challenge. No IL-1beta, TNF-alpha, IL-8 and C5a were detected using ELISA. mRNA of IL-1alpha, IL-1beta, IL-6, TNF-alpha, IL-8 and IL-12 were found in almost all the samples. The immunization triggered an early and massive neutrophil recruitment from the blood into the milk compartment as well as the recruitment of a cytotoxic/suppressor lyphocyte population during the early acute phase response. These results could help to devise new vaccinal strategies to fight against staphylococcal mastitis.