Neutrophils are implicated in many inflammatory lung disorders. However, the mechanisms regulating neutrophil migration in acute lung inflammation are incompletely understood. Although, integrin β2 mediates neutrophil migration in lungs in response to many stimuli such as E. coli, integrin involved in <i>S. pneumoniae</i> induced neutrophil migration is not known. Therefore, the role of integrin αvβ3 in neutrophil recruitment was tested. First, it was found that the number of neutrophils expressing the integrin subunits αv and β3 is reduced or remains in lung inflammation induced by E. coli or <i>S. pneumoniae</i>, respectively. Next, the role of integrin αvβ3 using β3 knockout mice (β3-/-) and function blocking antibodies was addressed. Neutrophil recruitment did not vary between wild type and β3-/- mice. Although β3 antibodies reduced neutrophil recruitment, similar effect was observed with isotype antibodies. Therefore, one can conclude that integrin αvβ3 is not critical for neutrophil recruitment in <i>S. pneumoniae</i> induced pneumonia. <p>Apart from integrins, TLR4 also regulate neutrophil migration. Because, the pattern of TLR4 expression at various times of lung inflammation is not known, TLR4 expression during different phases of lung inflammation in a rat model of LPS-induced inflammation was studied. TLR4 expression in the septum increased and decreased at 6h and 12-36h of inflammation, respectively. Since these correlate with the time of increase and decline of neutrophil recruitment, the findings support previously observed requirement for TLR4 in neutrophil recruitment. <p>Neutrophils recruited into the lungs regulate the inflammatory process by controlling subsequent monocyte/macrophage recruitment. The mechanisms involved and the pattern of monocyte/macrophage recruitment in lungs are not completely understood. Therefore, the possible involvement of monocyte chemoattractant protein (MCP)-1, which is a premier chemokine in monocyte/macrophage migration and produced by neutrophils and other cells was tested. This was addressed by quantification of monocytes/macrophages at various times and using neutrophil depletion experiments in LPS-induced lung inflammation in rats. It was found that monocytes/macrophages migrate very early and before neutrophils in addition to their migration in the late phase of acute lung inflammation. Neutrophil depletion abrogated both early as well as the late monocyte/macrophage recruitment without altering the expression of MCP-1. Therefore, possibly other chemokines and not MCP-1 are involved in neutrophil dependent monocyte/macrophage recruitment. <p>To conclude, the experiments further the understanding on acute lung inflammation by ruling-out the involvement of integrin αvβ3 and MCP-1 in β2-independent neutrophil migration and neutrophil dependent monocyte/macrophage recruitment, respectively. Further studies are essential to find the integrins and chemokines operating in the above situations. Equally important will be to understand the functional significance of early recruited monocytes/macrophages in the lung.

<p>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-1â, TNF-á, TGF-â 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-â and IL-10.</p><p>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-1â transcript levels between LPS challenged BDL rats and LPS challenged control rats (p=0.746) but ELISA showed increase in IL-1â concentration in LPS challenged BDL rats compared to LPS challenged control rats (p=0.000). TNF-á 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-1â and TNF-á 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-â 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. </p><p> 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-1â 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-1â 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-á 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-á 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-â 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-â concentrations. </p><p>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.</p>

<p>The success of implanted medical devices, such as biosensors, is dependent on the immune reaction to the surface of the implanted material. This immune reaction, termed the foreign body reaction, is potentially affected by the physical and chemical properties of the implanted material. Macrophages interact with the surface of the implanted material and secrete intercellular signals, including cytokines and growth factors, which direct the actions of immune cells in the surrounding tissue. The type and quantity of cytokines and growth factors produced by macrophages at an implant surface could be an indicator of the outcome of the foreign body reaction. </p><p>This study investigated the effect of the surface chemistry of an implanted device on the production of cytokines and growth factors. First, microdialysis sampling was characterized as a technique for collecting cytokines and growth factors from the tissue surrounding an implant. Based on this characterization, it was determined that a direct sampling method would be more suitable than microdialysis sampling for determining accurate tissue concentrations of cytokines and growth factors. Second, an in vitro model was developed and utilized to assess cytokine and growth factor production from monocyte/macrophage cultures seeded onto commonly implanted polymeric biomaterials with varying surface chemistries. The materials included in this study were polyethylene (PE), polyurethane (PU), polymethyl methacrylate (PMMA), expanded polytetrafluoroethylene (ePTFE), and a cytotoxic organo-tin polyvinyl chloride (ot-PVC) as a positive control. From this in vitro model, it was determined that the varying surface chemistries of these non-toxic materials, excluding ot-PVC, did not significantly affect the types and quantities of cytokines and growth factors produced. Finally, an in vivo model for evaluating the cytokine and growth factor response to an implanted biomaterial was utilized for comparison with the in vitro findings. In this model, biomaterials were implanted subcutaneously within the lumen of a stainless steel mesh cage. The mesh cage served to create a "pocket" where wound exudate fluid collected within the cage, surrounding the implanted biomaterial. The materials included in this study were PE, PU, and ot-PVC. Cytokines and growth factors produced at the material surface were sampled directly from the exudate fluid. The results from this in vivo study indicate that cytokine and growth factor production were not significantly impacted by the varying surface chemistries of the implanted biomaterials. The in vivo data support the findings from the in vitro model, suggesting that the foreign body reaction proceeds in a similar fashion for each of these non-cytotoxic, polymeric biomaterials with varying surface chemistries.</p> / Dissertation

[First published online] 2010-11-22 / 名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成23年3月25日 澤井晶穂氏の博士論文として提出された

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published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Human immunodeficiency virus (HIV) infection of macrophages in brain and other tissues plays an important role in development of HIV-associated neurological disorders and other aspects of disease pathogenesis. Macrophages express low levels of CD4, and macrophage-tropic HIV strains express envelope glycoproteins (Envs) adapted to overcome this restriction to virus entry by mechanisms that are not well characterized. One mechanism that influences this phenotype is increased exposure of the CD4 or CCR5 binding site, which may increase dissociation of soluble gp120 (sgp120) from Env trimers based on structural models. Little is known about spontaneous sgp120 shedding from primary HIV Envs or its biological significance. In this dissertation, we identify genetic determinants in brain-derived Envs that overcome the restriction imposed by low CD4, examine spontaneous sgp120 shedding by these Envs, and explore the biological significance of these findings. Sequence analysis of the gp120 beta-3 strand of the CCR5-binding site bridging sheet identified D197, which eliminates an N-linked glycosylation site, as a viral determinant associated with brain infection and HIV-associated dementia (HAD), and position 200 as a positively-selected codon in HAD patients. Mutagenesis studies showed that D197 and T/V200 enhance fusion and infection of macrophages and other cells expressing low CD4 by enhancing gp120 binding to CCR5. Sgp120 shedding from primary brain and lymphoid Envs was highly variable within and between patients, representing a spectrum rather than a categorical phenotype. Brain Envs with high sgp120 shedding mediated enhanced fusion and infection with cells expressing low CD4. Furthermore, viruses expressing brain Envs with high sgp120 shedding had an increased capacity to induce lymphocyte activation during PBMC infection, despite similar levels of viral replication. Genetic analysis demonstrated greater entropy and positive selection in Envs with high versus low levels of sgp120 shedding, suggesting that diversifying evolution influences gp120-gp41 association. Finally, we examined V3 loop sequences from dual-tropic brain and lymphoid Envs and found that the frequency of R5X4 HIV-1 is underestimated by most predictive bioinformatic algorithms. Together, these studies provide a better understanding of how neurotropic HIV Envs adapt to target cells expressing low CD4, and possible roles of these viral adaptations in disease pathogenesis.

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Les maladies inflammatoires du système nerveux central (SNC) sont caractérisées par l'altération de la barrière hémato-encéphalique induite par les cellules immunitaires et les cellules tumorales. Il est reconnu que les macrophages peuvent induire l'inflammation en infiltrant la BHE. Lors d'un dommage ou d'une infection du SNC, les macrophages dérivés du sang sont activés. Une fois activés, ceux-ci migrent au site infecté ou endommagé et libèrent des cytokines et médiateurs inflammatoires tels que IL1, TNFα, VEGF. Ces cytokines jouent un rôle essentiel dans l'inflammation du SNC puisqu'elles induisent des chimiokines tels que la sphingosine-1-phosphate (S1P), un sphingolipide fortement exprimé dans les glioblastomes et qui joue un rôle important dans la chimiotaxie et le trafic des cellules immunitaires. Nous avons étudié l'efficacité d'une molécule dérivée de notre diète possédant des propriétés chimiopréventives et anti-inflammatoires, l'épigallocatéchine gallate (EGCG), sur la régulation transcriptionnelle des récepteurs de la S1P à divers stades de différenciation des cellules promyélomonocytaires HL-60. Nous avons d'abord différencié les cellules promyélomonocytaires HL-60 en « macrophages-like » en utilisant un promoteur tumorigène et activateur de la protéine kinase C-Phorbol 12-myristate 13-acétate (PMA). Nous avons démontré que le PMA induit l'adhésion cellulaire et augmente l'expression des transcrits S1P1, S1P2 et S1P5. Nous avons ensuite constaté que les cellules adhérentes semblaient être sensibles à la S1P en induisant la phosphorylation d'ERK, de JNK et de P38 MAPK. Cependant, l'inclusion de l'EGCG avant la différenciation par le PMA inhibe l'induction de ces trois voies MAPK par la S1P. D'autre part, un traitement par l'EGCG au cours de la différenciation, c'est-à-dire simultanément avec le PMA, affecte seulement la voie P38 MAPK. De plus, les cellules différenciées « macrophages-like » devenaient insensibles à l'EGCG. Enfin, nous démontrons que seul le récepteur S1P2, parmi les récepteurs fortement induits par le PMA, diminue lors du prétraitement par l'EGCG. Nos résultats suggèrent que l'EGCG antagonise la réponse à la S1P dans les cellules prédifférenciées via l'inhibition de la signalisation induite par le PMA. Par conséquent, une réponse réduite à la S1P pourrait abroger la migration transendothéliale des monocytes vers le SNC, et prévenir la neuroinflammation, les infections cérébrales secondaires ou certaines pathologies cérébrales conséquentes à l'infiltration de cellules immunitaires. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Neuroinflammation, S1P, EGCG, macrophages, chimiotactisme, chimioprévention

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.