Year: 2005
Type: dissertation
Abstract: Humans with biliary cirrhosis are highly susceptible to acute pulmonary dysfunction and suffer from hepato-pulmonary syndrome.The mechanisms of this enhanced susceptibility remain unknown.It is well established that pulmonary intravascular macrophages (PIMs) are present in cattle, horses, goat and sheep and increase susceptibility for lung inflammation.Species such as rat and mouse also recruit PIMs especially in a bile duct ligation model of biliary cirrhosis.The contributions of recruited PIMs to lung inflammation associated with liver dysfunction remain unknown.Therefore, I characterized a bile duct ligation (BDL) model in rats to study role of recruited PIMs in lung inflammation.First, Sprague Dawley rats were subjected to BDL (N=6) or sham surgeries (N=3) and were euthanized at 4 weeks post-surgery.Five rats were used as the controls.Lung tissues were collected and processed for histology, immunohistology, immuno-electron microscopy, enzyme-linked immunosorbant assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR).Light microscopy demonstrated normal lung morphology in sham-operated and control rats but showed septal recruitment of mononuclear cells, which were positive for anti-rat monocytes/macrophage antibody ED-1, in BDL rats.(p=0.002).Immuno-electron microscopy confirmed localization of ED-1 in PIMs.BDL rats showed increased lung expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA compared to the controls (p=0.017)but not of IL-1β, TNF-α, TGF-β and IL-10.Then, I treated BDL rats (N=5) with gadolinium chloride (GC; 10 mg/Kg body weight intravenous) and found reduced numbers of PIMs (p=0.061) at 48 hours post-treatment along with increased expression of TGF-β and IL-10.I challenged control rats (N=5) and BDL rats (N=6) with Escherichia coli lipopolysaccharide (E. coli LPS; 0.1 mg/Kg body weight intravenous).All the BDL rats died within 3 hours of LPS challenge (100% mortality) while the normal LPS-treated rats were euthanized at 6 hours post-treatment.Histology and ED-1 staining showed dramatic increase in the number of septal monocytes/macrophages in BDL+LPS rats compared to normal LPS-treated rats (p=0.000).Staining of lung sections with an LPS antibody localized the LPS in lungs.RT-PCR analyses showed no differences in IL-1β transcript levels between LPS challenged BDL rats and LPS challenged control rats (p=0.746)but ELISA showed increase in IL-1β concentration in LPS iii challenged BDL rats compared to LPS challenged control rats (p=0.000).TNF-α mRNA (p=0.062) and protein (p=0.000) was increased in BDL+LPS rats compared to the control+LPS rats.Immuno-electron microscopy showed IL-1β and TNF-α in PIMs.BDL rats challenged with LPS showed increased expression of IL-10 mRNA and protein (p=0.000& 0.002 respectively) in lungs compared to LPS challenged control rats.TGF-β mRNA did not change (p=0.128)but lower protein concentrations (p=0.000) were observed in LPS-treated control rats compared to BDL+LPS.To further address the role of PIMs, I treated rats with GC at 6 hours or 48 hours (N=5 each) before LPS challenge.The mortality in the 6 hour group was 20% while all the rats in 48 hour group survived till 6 hours.Histology and ED-1 staining showed decrease in the number of intravascular cells in these groups compared to LPS treated BDL rats (p=0.039 for 6 hour group; p= 0.002 for 48 hour group).There were no differences in IL-1β mRNA in both 6 hour and 48 hour groups compared to the LPS challenged BDL rats (p=0.712& 0.509 respectively).ELISA showed no decrease in IL-1β concentration in 6 hour GC-treated group but a decrease was noticed at 48 hours compared to LPS challenged BDL rats (p=0.455& 0.008 respectively).TNF-α mRNA levels were not different between LPS-challenged GC-treated BDL rats and LPS-challenged BDL rats (p=0.499& 0.297 for 6 hour & 48 hour GC groups respectively).But TNF-α concentration in 48 hour GC group (p=0.001) but not in 6 hour GC group (p=0.572) was lower in comparison to BDL+LPS group.IL-10 mRNA was decreased in both 6 hour and 48 hour GC groups (p=0.038& 0.000 respectively) compared to LPS challenged BDL rats.ELISA showed decrease in IL-10 concentration in 48 hour GC group (p=0.030)but not in 6 hour GC group (p=0.420).TGF-β mRNA expression was decreased in 48 hour GC group (p=0.000)but not in 6 hour GC group (p=0.182).But GC treatment did not affect TGF-β concentrations.The data from these experiments characterize a BDL model to study PIM biology, show PIMs' pro-inflammatory potential and their possible role as a therapeutic target in lung inflammation.support, motivation, excellent guidance, friendly attitude and the time he devoted to me.Without his strong support, I would not have had the opportunity to undertake this study in the first place.I would also like to extend my sincere gratitude to my advisory committee members Dr. Tanya Duke and Dr. Gillian Muir for their guidance in my research project.I would also like to thank Sarah Caldwell, Jim Gibbons and the people at the Animal Care Unit for their excellent technical assistance.I would also like to thank Dr. Colette Wheler for helping me and giving me the valuable guidance during surgeries in rats.I am also
Author: Sukhjit Singh Gill
Language: English
Subfield: Hepatology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1514040099