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

Objective: To examine changes in inflammatory mediators, lymphocyte subpopu lations and neutrophil activation that occur during an immune-mediated recr uitment of neutrophils in the mammary gland. Subjects. 11 clinically healthy cows. Treatment: 5 cows received 2 subcutaneous injections of 30 mu g of alpha-to xin of Staphylococcus aureus, two months apart. Three months after the last immunization, 5 mu g of alpha-toxin were injected, via the teat end, in on e randomly selected quarter of the 5 immunized cows and of the 6 unimmunize d cows (control group). Methods: Blood and milk samples were collected at several times during 4 da ys post-challenge. Blood and milk cells were purified to be stained with sp ecific mAbs and analysed by flow cytometry, or to be used for cytokine RT-P CR. Bovine serum albumin, haptoglobin, cytokines and C5a were also analysed in milk or plasma samples using radial immunodiffusion assay or ELISA. Results. Large amounts of cells (>1 million/ml of milk) were recruited in t he quarters of the immunized cows, whereas no recruitment occurred in the c ontrol group. In blood of immunized animals, haptoglobin was present and ex pression of surface adhesion molecules on neutrophils was modified whereas no change was observed concerning the lymphocyte subpopulations. On milk-de rived neutrophils, the expression of CD11b and CD18 was upregulated compare d to blood, in contrast to CD62L that was downregulated. The CD8+ cells wer e recruited as soon as 12 h post-challenge, in contrast to the CD4+ cells, 96 h post-challenge. No IL-1 beta, TNF-alpha, IL-8 and C5a were detected us ing ELISA. mRNA of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, IL-8 and IL-12 w ere found in almost all the samples. Conclusions: The immunization triggered an early and massive neutrophil rec ruitment from the blood into the milk compartment as well as the recruitmen t of a cytotoxic/suppressor lymphocyte population during the early acute ph ase response. These results could help to devise new vaccinal strategies to fight against staphylococcal mastitis.