Induction of lgA secretion in the bovine mammary gland
Hodgkinson, A. J. (2005). Induction of lgA secretion in the bovine mammary gland (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/12923
Permanent Research Commons link: https://hdl.handle.net/10289/12923
This thesis investigates molecular and cellular mechanisms involved in lgA secretion in the bovine mammary gland. Our previous research showed that multi-site immunisation during late-pregnancy, involving priming by intra-peritoneal and intramuscular routes followed by intra-mammary immunisation, enhanced lgA secretion in milk and this effect was localised to immunised glands. This thesis addresses the hypothesis that increased lgA is produced by localised lgA plasma cells of mucosal origin that are recruited to the mammary gland by changes induced by intra-mammary immunisation. Recruitment mechanisms for lymphocytes were studied by examining expression of adhesion molecules MAdCAM-1, VCAM-1 and PNAd in mammary tissues of untreated animals, using immunohistochemical and molecular techniques. No immunoreactive MAdCAM-1 protein was found in venules and very low levels of MAdCAM-1 mRNA were found in tissues. By contrast, VCAM-1 protein was observed on larger venules. PNAd protein was only detected in supramammary lymph nodes, suggestive of a role for recruiting naïve lymphocytes. Cows were immunised with only one udder-half receiving intra-mammary immunisation to investigate effects on immunised glands. Animals with increased levels of lgA in milk had significantly more lgA plasma cells and T cells in immunised glands compared with non-immunised glands. Again no detectable MAdCAM-1 protein and only very low levels of MAdCAM-1 mRNA were found. No differences were observed in VCAM-1 protein expression between immunised and non-immunised mammary tissues. Together these studies suggest that MAdCAM-1 and VCAM-1 are not involved in lymphocyte recruitment to the bovine mammary gland, which contrasts to mice. In another study, cows categorised as either high- or low-responders to the multi-site immunisation were re-immunised to compare responses. Changes to lymphocytes in mammary secretions collected at intervals following two intra-mammary immunisations were monitored by flow cytometry. The percentage of T cells was found to decrease in both groups, although CD4 cells increased relative to CD8. The percentage of lgA-positive cells increased in a biphasic manner with high-responding animals showing an earlier and more sustained increase in lgA-positive cells compared with low-responders. The strong correlation observed between percentages of lgA-positive cells and lgA antibody in secretions suggests high levels of lgA require greater numbers of lgA-producing cells. Molecular analysis of known key cytokines found higher levels of IL-8 and IFN-𝛾 mRNA in immunised mammary tissues compared with non-immunised glands. In cells from mammary secretions, IL-8 mRNA increased shortly after immunisation while IFN- 𝛾 and TGF-β mRNA increased at a later stage. However, no discernible differences were observed between high- and low-responding animals. TNF-α, IL-2, IL-6 and IL-10 mRNA were found to be low in both mammary tissues and secretion cells. lgA mRNA was increased in immunised tissues although no correlation was observed between lgA mRNA and mRNA levels of the polyimmunoglobulin receptor. These analyses indicate that innate and cell-mediated responses were induced along with an antibody response to immunisation. Together these results support the hypothesis and indicate that intra-mammary immunisation increases lgA-producing cells in the mammary gland accompanied by changes in expression profiles of a range of cytokine genes. However, in contrast to mice, the data suggest little involvement of known adhesion molecules in recruiting lymphocytes to the gland.
The University of Waikato
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