| Summary: | 碩士 === 國立屏東科技大學 === 畜產系所 === 96 === Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) are among the most frequently isolated species of pathogens that induce bovine mastitis. In response to the invading pathogens, residential immune cells and mammary epithelail cells release chemoattractants to recruite neutrophils, which migrate from the blood into the milk to perform their bactericidal functions. Interleukin (IL)-8 and a recently identified granulocyte chemotactic protein (GCP-2) are the members of chemokines and their chemotactic effects on neutrophils have been demonstrated. However, the involvement of GCP-2 during the course of bovine mastitis has not been characterized. Therefore, the object of this study was to investigate the expression of GCP-2 and IL-8 in bovine neutrophils and mammary epithelial cells stimulated with various bacterial cell wall components, including lipoplysaccharide (LPS) from Gram-negative bacteria, lipoteichoic acid (LTA) and peptidoglycan (PGN) from Gram-positive bacteria. In addition, the chemotacic effect of recombinant human GCP-2 (rH-GCP-2) on bovine neutrophils was examined by using inserts cultured with a monolayer of bovine mammary epithelail cell line (MAC-T). The results showed that mammary epithelail cells increased the expression of both GCP-2 and IL-8 mRNA in response to different concentrations of LPS in a does-dependent manner. In the case of LTA, the expression of both chemokines was only significantly (P < 0.05) increased when the highest concentration was used (10 g/ml). PGN, at any concentrations tested, failed to increase the expression of both IL-8 and GCP-2. Therefore, among the three bacterial cell wall components, LPS is a much stronger stimulator in terms of inducing the expression of IL-8 and GCP-2 at the transcriptional level. On the other hand, the expression of IL-8 and GCP-2 in neutrophils was significantly increased when the lowest (1 ng/ml) concentration of LPS was used. LTA increased the expression of both chemokines at the highest concentration (1 g/ml). However, due to large variations, the differen- ces were not statistically significant. PGN had no effects on the expression of both IL-8 and GCP-2. Since recombinant bovine GCP-2 is not commercially available, we used rH-GCP-2 to characterize the chemotactic effect of GCP-2 on bovine neutorphils. In the assay, different concentrations of rH-GCP-2 (0, 1, 10, 100, 1000 ng/ml) were used to stimulate neutrphil migration for 3 h. The amount of migrated neutrophils, represented by the level of myeloperoxidase (MPO) activity, was slightly increased as the concentration of rH-GCP-2 increased. In a time-dependent assay, neutrohils were allowed to migrate for 3, 6, 12, and 24 h in response to 1 g/ml of rH-GCP-2. Results showed that the amount of migrated neutrophils was significantly increased as the time increased, indicating rH-GCP-2 is a weak chemoattractant for bovine neutrophils. The different responses of mammary epithelial cells to cell wall components from Gram-negative and Gram-positive bacteria might explain, at least in part, why mastitis induced by coliform and S. aureus has different properties and clinical symptoms.
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