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The lymphatic system provides a conduit for the trafficking of immune cells from the periphery to draining lymph nodes, both for constitutive immune surveillance and during inflammation. Thus, leucocyte migration into the lymphatics represents an important step in the initiation of a primary immune response, which occurs within lymph nodes. Traditionally, it has been considered that neutrophils are absent from the afferent lymph, having a finite lifespan in the periphery after extravasation from the blood. However, recent research has reported the presence of neutrophils in lymph nodes, in animal models of infection, where neutrophil trafficking was found to occur through afferent lymphatic vessels. This thesis examines the mechanisms regulating neutrophil migration, both by the lymphatic endothelium and neutrophils themselves, under resting and inflammatory conditions. Primary human dermal lymphatic endothelial cells (HDLECs) respond to the pro- inflammatory cytokine, TNF-α by instigating a distinct expression programme, characterised by the up-regulation of various adhesion molecules (ICAM-1, VCAM-1, E-selectin), and CXC- chemokines (ENA-78, GROβ, IL-8), as well as other potential regulators of neutrophil entry such as constitutively expressed adhesion molecules, CD99 and ESAM. Moreover, neutrophils possess counter-receptors for these adhesion molecules and contain basement membrane-specific proteases (elastase) and matrix metalloproteinases (MMPs) such as MMPs -8 and -9, localised in intracellular granules, ready to be exocytosed upon inflammatory stimuli. In vitro data presented in this thesis demonstrate that neutrophil adhesion and transmigration of the lymphatic endothelium is entirely dependent on prior activation of the monolayer with TNF-a. Furthermore, the aforementioned lymphatic-expressed adhesion molecules and chemokines, as well as neutrophil-derived proteases and MMPs are shown to play critical roles in neutrophil adhesion and transmigration of the lymphatic endothelium. Subsequent in vivo experiments confirmed that neutrophil trafficking to lymph nodes across lymphatic vessels is wholly dependent on prior vaccination with Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Tokyo-172. Moreover, neutrophils trafficking to lymph nodes across lymphatic vessels are shown to require ICAM-1. The results described in this thesis provide the first evidence that both the lymphatic endothelium and neutrophils act in concert to regulate entry to lymph nodes and determine the outcome of infection, or vaccination.

Copies of author's previously published articles inserted. / Bibliography: leaves 190-225. / vi, 225 leaves, [66] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the time course and degree of neutrophil polarisation in plasma and compares this response with those induced by FMLP, purified plasma proteins (particularly immunoglobulin type G) and chemotactic imflammatory mediators. In addition, the possible roles of extracellular divalent cations (Ca2+ and Mg2+), intracellular Ca2+ ions and actin microfilament distribution during responses to each stimulus are examined. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1993

Calcium (Ca$ sp{2+}$) and protein kinase C (PKC) are believed to act as intracellular signals triggering the activation of NADPH oxidase in neutrophils leading to superoxide generation. This was tested on bovine neutrophils by chelating extracellular and/or intracellular free Ca$ sp{2+}$ and by measuring PKC activity when the cells were stimulated by phorbol myristate acetate (PMA) or opsonized zymosan (OZ). Chelation of extracellular Ca$ sp{2+}$ with EGTA did not alter O$ sb2 sp{-}$ production from PMA stimulated cells. However, it did cause a 64% decrease in O$ sb2 sp{-}$ production in the neutrophils when stimulated with OZ. When intracellular Ca$ sp{2+}$ was chelated with BAPTA/AM, there was a significant decrease in O$ sb2 sp{-}$ generation following PMA activation. Yet, OZ activated cells, pre-treated with BAPTA/AM, showed an increase in the respiratory burst proportional to the chelator's concentration. Moreover, although OZ was previously shown to increase O$ sb2 sp{-}$ generation by neutrophils, no significant changes in PKC activity were observed. PMA stimulation led to an increase in PKC activity at the membrane level. Furthermore, treating the cells with calphostin C, a PKC activity inhibitor, caused a 69% decrease in O$ sb2 sp{-}$ production demonstrating the involvement of PKC in PMA-stimulated cells. However, no differences were observed between the OZ activated cells incubated with the inhibitor and the control cells. These data provide evidence that activation of NADPH oxidase can be achieved by either a PKC-dependent or a PKC-independent pathway depending on the stimulatory agent.

Neutrophil potential phagocytic capacity (NPPC), measured on 25 AI Canadian Holstein bulls, was investigated for evidence of association with production and type traits, SCC, and survival in dairy cows. Bulls were ranked based on different degrees of NPPC (Uptakes of 0, 1, 2, and 3 or more latex beads), using the solutions coming from an animal model. A total of 42,103 first lactation records, collected from 1985 through 1993 in 2,919 Quebec dairy herds, were used to obtain EBV's for SCC and for log SCC (LogSCC) for 697 sires. Correlations between NPPC measurements and somatic cell EBV's were null. Canadian official ETA's for type traits related with mammary system had a tendency of being positively correlated with higher NPPC and negatively with Uptake-0. Canadian official ETA's for production traits were negatively correlated with higher NPPC and positively with Uptake-0. A total of 17,202 first lactation records of daughters of the 25 AI bulls were used to study the effect of NPPC and log SCC on survival in dairy cows. Survival after first lactation was more closely related to sires' NPPC-EBV's than to LogSCC-EBV.

Diapedesis, phagocytosis and microbicidal activity are important parameters of neutrophil functionality and thus outcome of mastitis. An in vitro model of an "alveolar pavement" using the MAC-T3 bovine mammary epithelial cell line was developed to assess neutrophil diapedesis. Features of this biologically-meaningful barrier include: characteristic transepithelial resistance, tight junction complexes and polarity. Continuous transepithelial resistance measurements showed no significant changes throughout the assay period. Neither a Staphylococcus aureus challenge ($1 times10 sp7$ and $2 times10 sp9$ cfu/ml), or the presence of neutrophils, both resting and challenged had any deleterious effects on monolayer integrity over a short term (1-2 h) exposure. Neutrophils, both resting and challenged gave no indication of causing damage to the epithelium over the short term. Neutrophils isolated from proven sires and evaluated for phagocytic activity were found to differ significantly (p $<$ 0.05) in activity, rate and capacity to uptake particles. Correlations between phagocytic parameters and production traits were negative and small in magnitude. Microbicidal activity of neutrophils isolated from proven sires showed a highly significant variation between animals due to test day (p $<$ 0.001), however variation due to source of cells (i.e. animal) was not significant. in vitro analysis of diapedesis and phagocytosis is promising as a tool for the assessment of resistance or susceptibility to mastitis.

Bovine mastitis due to bacterial infection is one of the most costly diseases affecting the dairy industry. The polymorphonuclear neutrophils (PMNs) present in milk have a central protective role against invading pathogens, However, the manner by which PMNs traverse the secretory epithelia and the relationship between PMN diapedesis and the epithelial damage are unclear. This in vitro study investigated the process and rate of bovine PMN transepithelial migration. The bovine mammary epithelial cell line, MAC-T, formed a confluent monolayer with characteristic tight junctions, polarity and functional barrier to the dye trypan blue. In the first series of experiments, neutrophils were added into the upper compartment of the culture insert and stimulated to migrate across the epithelium in an apical-to-basal direction by the addition of Staphylococcus aureus to the lower compartment. Light and transmission electron microscopy revealed the following series of events for PMN transmigration: (1) adherence of PMNs to the surface of the epithelium; (2) projection of pseudopods toward the intercellular junction; (3) migration between adjacent epithelial cells; and (4) re-approximation of epithelial cell membranes and reformation.

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