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Understanding the immune defense of the mammary gland is important in devising and developing measures to control mastitis. Lipopolysaccharide (LPS) is the predominant factor causing pathogenesis in Gram-negative bacterial infections. The cluster of differential antigen14 (CD14), which is located on the surface of leukocytes, is the receptor for LPS. The binding of LPS to CD14 results in release of pro-inflammatory cytokines that recruit polymorphonuclear neutrophil leukocytes (PMNs), to the site of inflammation, allowing them to kill bacteria through the process of phagocytosis. This research was designed to produce and characterize monoclonal antibodies (mAb) against recombinant bovine soluble (rbos) CD14, express rbosCD14 in plants and characterize the biological activity of plant-derived rbosCD14 in vitro and in vivo. The release of inflammatory cytokines and expression of CD14 on bovine PMN and the secretion of IL-8 by PMN in response to LPS was also invesitigated.

      A panel of ten murine mAb reactive with rbosCD14 were produced and a sandwich ELISA was developed using the mAbs and rabbit polyclonal antibodies. The mAbs recognized rbosCD14 (40 kDa), solubleCD14 (sCD14) (53 and 58 kDa) in milk and blood, and a 47 kDa mCD14 in lysates of macrophages obtained from involuted bovine mammary gland secretions by Western blot analysis.  Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions.  The addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced TNF-alpha production in vitro.  The anti-rbosCD14 mAbs generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretion produced during mastitis.

        This study demonstrates the functional activity of a recombinant animal receptor protein made in plants, and the use of a plant-derived protein as a potential animal therapeutic for bacterial infections.  A truncated form of sCD14, carrying a histidine residue affinity tag was incorporated with potato virus X for transient expression in Nicotiana benthamiana.  CD14 from crude plant extracts was recognized by Western blot analysis.  Biological activities of plant-derived rbosCD14 (prbosCD14) were characterized in vitro and in vivo.  Biological activity of prbosCD14 demonstrated in vitro by LPS-induced apoptosis, an increase of caspase activity and IL-8 production by bovine endothelial cells.  In vivo, prbosCD14 enhanced LPS-induced PMN recruitment and in bovine mammary quarters challenged with Escherichia coli that resulted in decreased bacterial counts and elimination of clinical symptoms.

In this disseration, shedding of sCD14 from the surface of PMN in response to LPS was negatively correlated with IL-8 release.  Shedding of sCD14 from the surface of PMN increased in the absence of serum and at high concentrations of LPS.  The use of real time RT-PCR showed that CD14 gene expression was not different between control and the LPS stimulated cells.  This demonstrates that shedding of sCD14 comes from membrane-boundCD14 (mCD14).  However, in contrast to the shedding of sCD14, IL-8 secretion by PMN was decreased at high concentrations of LPS in the absence of serum.  Moreover, sCD14 secretion from PMN stimulated with LPS was increased in parallel with the decrease of IL-8 secretion at varying PMN cell densities.  This result suggests that the release of CD14 leads to the down-regulation of IL-8 secretion by PMN in response to LPS.

Bovine PMN produced different types of cytokines in response to LPS in this disseration. The secretion of TNF-alpha, IL-1beta, IL-8 and INF-gamma by PMN increased in a dose-dependent manner, but IL-12 secretion by PMN was decreased with increasing concentrations of LPS. Co-incubation of LPS with either TNF-alpha or IL-1beta increased secretion of IL-8 when compared to LPS alone.

These studies contribute to a better understanding of the interrelationship between bovine CD14 and LPS during mastitis and provide new insights into events that resolve inflammation following bovine PMN activation.