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Monocytes/macrophages play very important roles in innate and adaptive immunity. Ion channels are small molecules embedded in the cell membrane and they play fundamental roles in cell function. Both topics have been extensively studied in isolation, however the role of ion channels in macrophage function is far less understood. In this thesis, the functional and molecular aspects of two ion channels expressed in macrophages, Kor, a potassium channel, and CLIC1, a chloride channel were studied. The biological function of Kor and CLIC1 in activated human macrophages was examined using ion channel blockers. In addition, the role of CLIC1 in the cell cycling of CHO-K1 cells was also investigated. The in vitro studies showed that Kor and CLIC1 are involved in cytokine production by PMA-activated human macrophages and that CLIC1 is also involved in the cell cycling of CHO-K1 cells. Despite providing interesting data, the results of the in vitro studies were difficult to interpret due to the uncertain specificities of the Cl- channel blockers. Therefore, to understand the biological role of CLIC1 in vivo, a gene targeting experiment was performed to create a CLIC1 knock-out (KO) mouse. This involved cloning the mouse CLIC1 gene, making a targeting vector, producing targeted ES cells, and generating a CLIC1 knock-in (KI) mouse which carries a flag tag at the N-terminal and three loxP sites in the targeted locus. Crossing the CLIC1 KI mouse with the TNAP-Cre mouse, a strain over expressing Cre recombinase under a TNAP promoter, a CLIC1 KO mouse was generated. The initial phenotype analysis showed no major development or growth abnormality in the CLIC1 KO mouse. Instead, hyperplasia of megakaryocytes and possible erythroid cells in the spleen and bone marrow was observed suggesting some degree of abnormality in the haematopoeitic system. Furthermore, a comparison of wild type mice with the CLIC1 KO mouse showed that CLIC1 protein expresses at high levels in monocytes, lymphocytes, platelets, and tissue macrophages of normal animals tissues, such as spleen, kidney (mesangial cell), and liver (kupffer cells). This further indicates that CLIC1 may play a significant role in regulating functions of platelets, lymphocytes, and specially tissue macrophages. More extensive studies can now be performed on the CLIC1 KO mouse to clarify the biological function of CLIC1. In summary, the generation of the CLIC1 KO mouse provides a valuable model to study the biological function of CLIC1 both in vivo and in vitro.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1a, IL-1b, IL-6, IL-8 and TNF-a were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1α, IL-1β, IL-6, IL-8 and TNF-α were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

Legionella pneumophila (Lp) is the pathogen responsible for Legionnaires disease. Lp invades and survives in human macrophages to form an intracellular compartment, called the Legionella containing vacuole (LCV). Within the LCV, Lp avoids degradation and replicates, before killing its host and releasing its virulent progeny. Lp is pleomorphic, exhibiting a combination of short rod phenotypes and filaments. Filamentous Lp has been reported in patient samples, but current studies of Lp virulence are exclusively focused on rods. Our results show that filamentous Lp can invade and replicate in human and murine macrophages. Filaments are phagocytosed gradually into tubular phagocytic cups. Interestingly, the formation of the LCV starts at this stage of phagocytosis, and LCV markers were detected before the sealing of the phagosome occurred. We present evidence that the filamentous morphology acts cooperatively with Lp effectors to subvert the microbicidal activities of the macrophage, contributing to the survival of Lp.

Legionella pneumophila (Lp) is the pathogen responsible for Legionnaires disease. Lp invades and survives in human macrophages to form an intracellular compartment, called the Legionella containing vacuole (LCV). Within the LCV, Lp avoids degradation and replicates, before killing its host and releasing its virulent progeny. Lp is pleomorphic, exhibiting a combination of short rod phenotypes and filaments. Filamentous Lp has been reported in patient samples, but current studies of Lp virulence are exclusively focused on rods. Our results show that filamentous Lp can invade and replicate in human and murine macrophages. Filaments are phagocytosed gradually into tubular phagocytic cups. Interestingly, the formation of the LCV starts at this stage of phagocytosis, and LCV markers were detected before the sealing of the phagosome occurred. We present evidence that the filamentous morphology acts cooperatively with Lp effectors to subvert the microbicidal activities of the macrophage, contributing to the survival of Lp.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1α, IL-1β, IL-6, IL-8 and TNF-α were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1a, IL-1b, IL-6, IL-8 and TNF-a were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 43-65).

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references.