Glucans are (1-3)-β-D-linked polymers of glucose that are produced as fungal cell wall constituents and are also released into the extracellular milieu. Glucans modulate immune function via macrophage participation. The first step in macrophage activation by (1-3)-β-D-glucans is thought to be the binding of the polymer to specific macrophage receptors. We examined the binding/uptake of a variety of water soluble (1-3)-β-D-glucans and control polymers with different physicochemical properties to investigate the relationship between polymer structure and receptor binding in the CR3- human promonocytic cell line, U937. We observed that the U937 receptors were specific for (1→3)-β-D-glucan binding, since mannan, dextran, or barley glucan did not bind. Scleroglucan exhibited the highest binding affinity with an IC50 of 23 nM, three orders of magnitude greater than the other (1→3)-β-D-glucan polymers examined. The rank order competitive binding affinities for the glucan polymers were scleroglucan >>> schizophyllan > laminarin > glucan phosphate > glucan sulfate. Scleroglucan also exhibited a triple helical solution structure (ν = 1.82, β = 0.8). There were two different binding/uptake sites on U937 cells. Glucan phosphate and schizophyllan interacted nonselectively with the two sites. Scleroglucan and glucan sulfate interacted preferentially with one site, while laminarin interacted preferentially with the other site. These data indicate that U937 cells have at least two non-CR3 receptor(s) which specifically interact with (1→3)-β-D-glucans and that the triple helical solution conformation, molecular weight and charge of the glucan polymer may be important determinants in receptor ligand interaction.

The a7 nicotinic acetylcholine receptor (a7nAChR) expressed on macrophages, a critical link between the neural and immune systems, provides a defense mechanism during inflammatory diseases. Release of acetylcholine in target tissues activates a7nAChR, a necessary element of the cholinergic anti-inflammatory pathway, inhibiting pro-inflammatory signaling pathways via NF-kB. However, other potential aspects of a7nAChR function are not well understood. The purpose of our project is to evaluate the role of a7nAChR activation in macrophage migration and accumulation at the site of inflammation. We first evaluated the survival of WT and a7nAChR-/- mice during LPS-induced endotoxemia. We found that a7nAChR-/- mice have significantly decreased survival compared to WT. We next examined differences in monocyte migration by tracking adoptively transferred, fluorescently labeled a7nAChR-/- and WT monocytes in the model of LPS-induced endotoxemia using flow cytometry and imaging flow cytometry. We found that a7nAChR-/- monocytes have significantly reduced migration to the lung, liver, and spleen during endotoxemia, in both a7nAChR-/- and WT recipient mice. Based on this result, we investigated if adoptive transfer of WT or a7nAChR-/- monocytes would decrease or improve survival in LPS-induced endotoxemia. Adoptively transferred, unlabeled WT monocytes improved survival in a7nAChR-/- recipient mice, though this effect did not reach significance. Survival was unaffected by adoptive transfer of a7nAChR-/- monocytes. Notably, this data coincides with the protective role of both macrophages and a7nAChR during sepsis that has been demonstrated in multiple recent publications. To reveal a potential mechanism, we tested the effect of a7nAChR-deficiency on the expression of adhesive and chemokine receptors at the macrophage surface. We selected 10 receptors to evaluate via qRT-PCR and flow cytometry. Our qRT-PCR experiments demonstrated a significantly reduced expression in CCR5, CCR2, integrin αMß2, and integrin αXß2 in a7nAChR-/- peritoneal macrophages, when compared to WT. The reduction in expression of CCR2 and αXß2 was corroborated by our flow cytometry results. Interestingly, the decrease in CCR5 and αMß2 was lower, but still detectable, and this discrepancy can be attributed to post transcriptional regulation of these receptors. The role of these receptors was further investigated in an in vitro 3D migration assay. Macrophages deficient in a7nAChR showed significantly decreased migration within a fibrin matrix (integrin αMß2 dependent) along a RANTES gradient (CCR5-mediated). The reduction in the migration of a7nAChR-/- macrophages toward MCP-1 (CCR2-mediated) did not reach significance, although still measurable. This experiment confirmed the chemokine-independent contribution of αMß2 to mesenchymal macrophage migration. These protective effects of αMß2 and CCR5 during sepsis were reported previously and are related to the regulation of monocyte recruitment and efflux at the site of inflammation. In summary, we discovered a new link between the parasympathetic nervous system and immune response based on a7nAChR-regulated macrophage migration during inflammation. The signaling pathway downstream of a7nAChR that modulates αMß2 and CCR5 expression is yet to be identified and is the objective of our ongoing investigation

The a7 nicotinic acetylcholine receptor (a7nAChR) expressed on macrophages, a critical link between the neural and immune systems, provides a defense mechanism during inflammatory diseases. Release of acetylcholine in target tissues activates a7nAChR, a necessary element of the cholinergic anti-inflammatory pathway, inhibiting pro-inflammatory signaling pathways via NF-kB. However, other potential aspects of a7nAChR function are not well understood. The purpose of our project is to evaluate the role of a7nAChR activation in macrophage migration and accumulation at the site of inflammation. We first evaluated the survival of WT and a7nAChR-/- mice during LPS-induced endotoxemia. We found that a7nAChR-/- mice have significantly decreased survival compared to WT. We next examined differences in monocyte migration by tracking adoptively transferred, fluorescently labeled a7nAChR-/- and WT monocytes in the model of LPS-induced endotoxemia using flow cytometry and imaging flow cytometry. We found that a7nAChR-/- monocytes have significantly reduced migration to the lung, liver, and spleen during endotoxemia, in both a7nAChR-/- and WT recipient mice. Based on this result, we investigated if adoptive transfer of WT or a7nAChR-/- monocytes would decrease or improve survival in LPS-induced endotoxemia. Adoptively transferred, unlabeled WT monocytes improved survival in a7nAChR-/- recipient mice, though this effect did not reach significance. Survival was unaffected by adoptive transfer of a7nAChR-/- monocytes. Notably, this data coincides with the protective role of both macrophages and a7nAChR during sepsis that has been demonstrated in multiple recent publications. To reveal a potential mechanism, we tested the effect of a7nAChR-deficiency on the expression of adhesive and chemokine receptors at the macrophage surface. We selected 10 receptors to evaluate via qRT-PCR and flow cytometry. Our qRT-PCR experiments demonstrated a significantly reduced expression in CCR5, CCR2, integrin αMß2, and integrin αXß2 in a7nAChR-/- peritoneal macrophages, when compared to WT. The reduction in expression of CCR2 and αXß2 was corroborated by our flow cytometry results. Interestingly, the decrease in CCR5 and αMß2 was lower, but still detectable, and this discrepancy can be attributed to post transcriptional regulation of these receptors. The role of these receptors was further investigated in an in vitro 3D migration assay. Macrophages deficient in a7nAChR showed significantly decreased migration within a fibrin matrix (integrin αMß2 dependent) along a RANTES gradient (CCR5-mediated). The reduction in the migration of a7nAChR-/- macrophages toward MCP-1 (CCR2-mediated) did not reach significance, although still measurable. This experiment confirmed the chemokine-independent contribution of αMß2 to mesenchymal macrophage migration. These protective effects of αMß2 and CCR5 during sepsis were reported previously and are related to the regulation of monocyte recruitment and efflux at the site of inflammation. In summary, we discovered a new link between the parasympathetic nervous system and immune response based on a7nAChR-regulated macrophage migration during inflammation. The signaling pathway downstream of a7nAChR that modulates αMß2 and CCR5 expression is yet to be identified and is the objective of our ongoing investigation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19571号 / 医博第4078号 / 新制||医||1013(附属図書館) / 32607 / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 武藤 学, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Previous studies have suggested that T cell contact‐dependent signaling of macrophages (MΦ) is mediated by membrane tumor necrosis factor‐α (memTNF‐α), based on the observation that anti‐TNF‐α could inhibit T cell‐mediated MΦ activation. The current report confirms that anti‐TNF‐α does inhibit activation of interferon‐γ (IFN‐γ)‐primed MΦ by paraformaldehyde‐fixed activated T cells. However, the involvement of membrane molecules other than memTNF‐α in the contact‐dependent signaling is suggested by two lines of evidence. First, the TH2 clone, AK8, displayed neither secreted TNF‐α/β nor memTNF‐α/β detectable by bioassay or immunofluorescence. Nonetheless, AK8 cells were equally effective, on a per cell basis, in contact‐dependent signaling of MΦ activation as TH2 and TH1 cells which do express memTNF‐α. Second, the expression of memTNF‐α by the TH clone, D10.G4, is maximal 24 h after activation, whereas the ability of this clone to activate MΦ is maximal at 6–8 h of activation and declines thereafter. Since TNF‐α is known to play a critical role in activation of MΦ effector function, it was hypothesized that T cell membrane components other than memTNF‐α might signal MΦ production of TNF‐α, thus allowing autocrine TNF‐α stimulation of MΦ effector function. In support of this, it is demonstrated that paraformaldehyde‐fixed activated TH2 cells can induce de novo production and release of TNF‐α by MΦ. This effect was not an artifactual result of paraformaldehyde fixation since paraformaldehyde‐fixed resting T cells did not induce TNF‐α gene expression. Previous studies have demonstrated a role for autocrine TNF‐α stimulation in LPS induction of effector function in recombinant IFN‐γ‐primed MΦ. The current study confirms that TNF‐α plays a critical role in T cell contact‐dependent signaling of MΦ but indicates that memTNF on the T cells may not be a sine qua non factor for contact‐dependent signaling. The data suggest that other T cell membrane molecules contribute to activation of MΦ effector function by stimulation of MΦ TNF‐α production.

No description available.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Neuroinflammation has been linked to the pathogenesis of many diseases, including chronic traumatic encephalopathy (CTE). CTE is a progressive neurodegenerative disease caused by exposure to repeated head impacts (RHI) from a variety of sources, including contact sports and military injury. CTE is characterized neuropathologically by the deposition of hyperphosphorylated tau (p-tau) in neurons as neurofibrillary tangles (NFT) and neurites at the depths of the cortical sulci in an irregular pattern. In addition to p-tau accumulation, there is also an accumulation of pigment-containing macrophages around small blood vessels in the white matter and widespread microglial inflammation in CTE. Macrophage and microglial inflammation can be beneficial to tissue repair, but if persistent, can precipitate neurodegeneration. This study quantified the density of perivascular CD68+ macrophages in the dorsolateral prefrontal (DLF) white matter, a brain region known to be affected early in CTE in post-mortem brain tissue from 46 individuals, 7 controls (mean age: 46.14, SD: 11.39, range: 22-55), 20 individuals exposed to RHI without CTE (mean age: 22.75, SD: 3.65, range: 17- 29), and 19 individuals, all of them American football players, with pathologically verified CTE (mean age: 25.11, SD: 2.92, range: 18-29). Brain tissue was provided by the Injury and Traumatic Encephalopathy (UNITE) brain bank and the post-traumatic stress disorder (PTSD) brain bank. Comparisons were made between controls, individuals exposed to RHI without CTE, and individuals with CTE. Fixed tissue samples of the DLF cortex and white matter were cut at 10μm and stained with CD68 to mark perivascular macrophages. Slides were imaged with a brightfield microscope at 40x magnification and analyzed using the HALO image software analysis platform. In the total population, a one-way test of variance (ANOVA) revealed a statistically significant increase in perivascular macrophages, indicated by CD68 positive pixels, in Stage III CTE compared to controls (p<0.05), a significant increase in Stage II compared to Stage I CTE (p<0.05), and a statistically significant increase in Stage III compared to Stage I CTE (p<0.01). The analysis also revealed a trend toward more CD68 pixels in Stage II CTE compared to controls (p=0.0883) and a trend toward more pixels in Stage III CTE compared to RHI no CTE (p=0.0705). Among the American football players, analyses revealed that Stage II CTE had significantly more perivascular macrophages than Stage I CTE (p<0.01), Stage II CTE had significantly more than controls (p<0.01), Stage III CTE had significantly more than Stage I CTE (p<0.05), and Stage III CTE had significantly more than controls (p<0.05). In summary, this study demonstrates that there is an increase in perivascular CD68 positive macrophages in individuals exposed to RHI with and without CTE. Perivascular macrophages and other neuroinflammatory molecules may play a critical role in the pathogenesis of CTE.

The essential nutrient choline is known to serve as a precursor for phospholipids and the neurotransmitter acetylcholine, and to feed into methylation pathways. The role and fate of choline in immune cells, however, is not yet fully elucidated. To act as a methyl group donor, choline must first undergo oxidation in mitochondria, a process which has long been thought to occur exclusively in the liver and kidney. The recent identification of choline transporters on the mitochondrial membrane has highlighted the possibility of mitochondrial choline oxidation in other cell types. Here, I show that choline transporters are present on the mitochondrial membrane of primary and immortalized mouse macrophages. The interaction of CTL2 with mitochondria is further augmented following pro-inflammatory polarization with the bacterial endotoxin lipopolysaccharide. I show that mitochondrial choline uptake occurs in macrophages using radiolabelled choline assays; however, it remains unclear whether this process is conducted through the identified transporters. Preliminary data suggest that mitochondrial choline oxidation to betaine was increased in LPS-stimulated macrophages, revealing a potential additional input into one-carbon metabolism in polarized macrophages. This project broadens the existing paradigm that choline oxidation occurs strictly in hepatic and renal tissue and suggests that choline oxidation may be a regulated process in macrophage polarization.

No description available.