Axon Guidance Molecules Guiding Neuroinflammation

Axon guidance molecules (AGMs), such as Netrins, Semaphorins, and Ephrins, have long been known to regulate axonal growth in the developing nervous system. Interestingly, the chemotactic properties of AGMs are also important in the postnatal period, such as in the regulation of immune and inflammatory responses. In particular, AGMs play pivotal roles in inflammation of the nervous system, by either stimulating or inhibiting inflammatory responses, depending on specific ligand-receptor combinations. Understanding such regulatory functions of AGMs in neuroinflammation may allow finding new molecular targets to treat neurodegenerative diseases, in which neuroinflammation underlies aetiology and progression.

Acute Phase Protein Lipocalin-2 Is Associated with Formalin-induced Nociception and Pathological Pain

Lipocalin-2 (LCN2) is an acute-phase protein induced by injury, infection, or other inflammatory stimuli. LCN2 binds small hydrophobic ligands and interacts with cell surface receptor to regulate diverse cellular processes. The role of LCN2 as a chemokine inducer in the central nervous system (CNS) has been previously reported. Based on the previous participation of LCN2 in neuroinflammation, we investigated the role of LCN2 in formalin-induced nociception and pathological pain. Formalin-induced nociceptive behaviors (licking/biting) and spinal microglial activation were significantly reduced in the second or late phase of the formalin test in Lcn2 knockout mice. Likewise, antibody-mediated neutralization of spinal LCN2 attenuated the mechanical hypersensitivity induced by peripheral nerve injury in mice. Taken together, our results suggest that LCN2 can be therapeutically targeted, presumably for both prevention and reversal of acute inflammatory pain as well as pathological pain.

Modulation of Glial and Neuronal Migration by Lipocalin-2 in Zebrafish

BACKGROUND: Glial cells are involved in immune and inflammatory responses in the central nervous system (CNS). Glial cells such as microglia and astrocytes also provide structural and functional support for neurons. Migration and morphological changes of CNS cells are associated with their physiological as well as pathological functions. The secreted protein lipocalin-2 (LCN2) has been previously implicated in regulation of diverse cellular processes of glia and neurons, including cell migration and morphology. METHODS: Here, we employed a zebrafish model to analyze the role of LCN2 in CNS cell migration and morphology in vivo. In the first part of this study, we examined the indirect effect of LCN2 on cell migration and morphology of microglia, astrocytes, and neurons cultured in vitro. RESULTS: Conditioned media collected from LCN2-treated astrocytes augmented migration of glia and neurons in the Boyden chamber assay. The conditioned media also increased the number of neuronal processes. Next, in order to further understand the role of LCN2 in the CNS in vivo, LCN2 was ectopically expressed in the zebrafish spinal cord. Expression of exogenous LCN2 modulated neuronal cell migration in the spinal cord of zebrafish embryos, supporting the role of LCN2 as a cell migration regulator in the CNS. CONCLUSION: Thus, LCN2 proteins secreted under diverse conditions may play an important role in CNS immune and inflammatory responses by controlling cell migration and morphology.

Cell to Cell Interaction Can Activate Membrane-bound APRIL Which Are Expressed on Inflammatory Macrophages

BACKGROUND: APRIL, originally known as a cytokine involved in B cell survival, is now known to regulate the inflammatory activation of macrophages. Although the signal initiated from APRIL has been demonstrated, its role in cellular activation is still not clear due to the presence of BAFF, a closely related member of TNF superfamily, which share same receptors (TACI and BCMA) with APRIL. METHODS: Through transfection of siRNA, BAFF-deficient THP-1 cells (human macrophage-like cells) were generated and APRIL-mediated inflammatory activities were tested. The expression patterns of APRIL were also tested in vivo. RESULTS: BAFF-deficient THP-1 cells responded to APRIL-stimulating agents such as monoclonal antibody against APRIL and soluble form of TACI or BCMA. Furthermore, co-incubation of the siBAFF-deficient THP-1 cells with a human B cell line (Ramos) resulted in an activation of THP-1 cells which was dependent on interactions between APRIL and TACI/BCMA. Immunohistochemical analysis of human pathologic samples detected the expression of both APRIL and TACI in macrophage-rich areas. Additionally, human macrophage primary culture expressed APRIL on the cell surface. CONCLUSION: These observations indicate that APRIL, which is expressed on macrophages in pathologic tissues with chronic inflammation, may mediate activation signals through its interaction with its counterparts via cell-to-cell interaction.

Antitumor Effects and Immunomodulating Activities of Phellinus linteus Extract in a CT-26 Cell-Injected Colon Cancer Mouse Model

The antitumor effects of Phellinus linteus extract (Keumsa Linteusan) were investigated in a CT-26 cell-injected colon cancer mouse model. When administered orally (250~1,000 mg/kg body weight), Keumsa Linteusan significantly inhibited the growth of solid colon cancer. The highest dose was highly effective, reducing tumor formation by 26% compared with the control group. The anticomplementary activity of Keumsa Linteusan increased in a dose-dependent manner. Lysosomal enzyme activity of macrophages was increased by 2-fold (100 microg/ml) compared with the control group. Keumsa Linteusan can be regarded as a potent enhancer of the innate immune response, and can be considered as a very promising candidate for antitumor action.

The Stimulation of CD147 Induces MMP-9 Expression through ERK and NF-kappaB in Macrophages: Implication for Atherosclerosis

BACKGROUND: CD147, as a cellular receptor for cyclophilin A (CypA), is a multifunctional protein involved in tumor invasion, inflammation, tissue remodeling, neural function, and reproduction. Recent observations showing the expression of CD147 in leukocytes indicate that this molecule may have roles in inflammation. METHODS: In order to investigate the role of CD147 and its ligand in the pathogenesis of atherosclerosis, human atherosclerotic plaques were analyzed for the expression pattern of CD147 and CypA. The cellular responses and signaling molecules activated by the stimulation of CD147 were then investigated in the human macrophage cell line, THP-1, which expresses high basal level of CD147 on the cell surface. RESULTS: Staining of both CD147 and CypA was detected in endothelial cell layers facing the lumen and macrophage-rich areas. Stimulation of CD147 with its specific monoclonal antibody induced the expression of matrix metalloproteinase (MMP)-9 in THP-1 cells and it was suppressed by inhibitors of both ERK and NF-kappaB. Accordingly, the stimulation of CD147 was observed to induce phosphorylation of ERK, phosphorylation-associated degradation of IkappaB, and nuclear translocation of NF-kappaB p65 and p50 subunits. CONCLUSION: These results suggest that CD147 mediates the inflammatory activation of macrophages that leads to the induction of MMP-9 expression, which could play a role in the pathogenesis of inflammatory diseases such as atherosclerosis.

LIGHT is Expressed in Foam Cells and Involved in Destabilization of Atherosclerotic Plaques through Induction of Matrix Metalloproteinase-9 and IL-8

BACKGROUND: LIGHT (TNFSF14) is a member of tumor necrosis factor superfamily and is the ligand for TR2 (TNFRSF14/HVEM). LIGHT is known to have pro- inflammatory roles in atherosclerosis. METHODS: To find out the expression pattern of LIGHT in atherosclerotic plaques, immunohistochemical analysis was performed on human carotid atherosclerotic plaque specimens. LIGHT induced atherogenic events using human monocytic cell line THP-1 were also investigated. RESULTS: Immunohistochemical analysis revealed expression of LIGHT and TR2 in foam cell rich regions in the atherosclerotic plaques. Double immunohistochemical analysis further confirmed the expression of LIGHT in foam cells. Stimulation of THP-1 cells, which express TR2, with either recombinant LIGHT or immobilized anti-TR2 monoclonal antibody induced interleukin-8 and matrix metalloproteinase(MMP)-9. Electrophoretic mobility shift assay demonstrated that LIGHT induces nuclear localization of transcription factor, nuclear factor (NF)-kappaB. LIGHT induced activation of MMP-9 is mediated by NF-kappaB, since treatment of THP-1 cells with the NF-kappaB inhibitor PDTC (pyrrolidine dithiocarbamate) completely blocked the activation of MMP-9. CONCLUSION: These data indicate that LIGHT is expressed in foam cells in atherosclerotic plaques and is involved in atherogenesis through activation of pro-atherogenic cytokine IL-8 and destabilization of plaque by inducing matrix degrading enzyme.

Cardiac Hypertrophy in Transgenic Mice with Overexpressed Small-Subunit of Human Myosin Light Chain Phosphatase

BACKGROUND AND OBJECTIVES: Recent reports have demonstrated that perturbation of the balance between myo-sin light chain (MLC) phosphorylation and the dephosphorylation status is associated with the development of cardiac hypertrophy. Myosin light chain phosphatase (MLCP) is a key enzyme that regulates the phosphorylation status of the MLC, but its functional roles in cardiac muscle have not been well investigated. Especially, the functions of the small-subunit of MLCP in cardiac muscles are not well elucidated. Here, whether the human heart-specific small-subunit (M21) of MLCP is associated with hypertrophic responses in a transgenic mice model were assessed. MATERIALS AND METHODS: The transgenic mice, overexpressing the human M21, were generated from a cardiac-specific transgenic construct. Cardiac tissues from the transgenic mice were subjected to histology for their morphological examination. The echocardiographic parameters of the murine heart were examined with transgenic mice aged 1, 2 and 3 months, and compared with their non-transgenic littermates. To determine whether the transgenic heart was sensitive to stress, the echocardiographic examination was also performed at the baseline, both before and after the administration of isoproterenol, at a dosage of 30 microgram/g/day, for 2 weeks. RESULTS: The histological analysis of the transgenic heart revealed myocyte disarray and nuclear hypertrophy. No significant differences were observed between the transgenic and non-transgenic mice in relation to the echocardiographic determinants, such as the left ventricular dimensions and the wall thickness. Chronic cardiac stress, induced by isoproterenol infusion, also failed to show any significant differences in relation to the same determinants. CONCLUSION: Overexpression of the human M21 in the murine heart induced myocyte hypertrophy. However, the overall cardiac functions were not affected under normal and stressed conditions.

Plasma Inflammatory Markers and T-Lymphocytes Are Activated in Angina Pectoris

BACKGROUND AND OBJECTIVES: Inflammation and activation of immune cells play important roles in the pathogenesis of atherosclerosis. We investigated the activation status of plasma inflammatory markers and immune cells in angina patients. METHODS: We analyzed the plasma level of C-reactive protein (CRP) as a marker of inflammation in 24 patients with angina pectoris (12 unstable angina, 12 stable angina), and 12 normal subjects. The degree of activation of peripheral blood monocytes was assessed by Northern analysis of pro-atherogenic cytokines and the activation status of T-lymphocytes was measured by flow-cytometric analysis of HLA-DR expression on T-cells. RESULTS: Plasma level of CRP was highest in unstable angina patients (1.63+/-0.70 mg/l) and lowest in the control subjects (0.22+/-0.08 mg/l)(p=0.03). We also observed a high correlation between CRP level and the occurrence of minor and major coronary events during 6 months of follow-up. The percentage of HLA-DR positive T-lymphocyte was significantly increased in the unstable angina patients (26.8+/-1.4%) compared with that in the control (14.7+/-1.2%)(p=0.0053). When baseline levels of cytokine mRNA were measured in monocytes, the percentages of the patients expressing higher than normal levels of IL-8, IL-1b, MCP-1, and TF mRNAs was 37.5, 29.2, 33.3, and 37.5%, respectively (p=0.0143, 0.0371, 0.0233, and 0.0143, respectively). Basal mRNA levels of interleukin (IL)-8, tissue factor (TF), IL-1b and monocyte chemoattractant protein-1 (MCP-1) showed a strong correlation with each other (p<0.01 in all combination) but not with tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta1. CONCLUSION: We observed increase in plasma CRP levels and activation of T-lymphocytes in angina patients. These results may help further classification of angina patients according to the activation of inflammatory markers and understanding the prognosis of the disease.

Aging affects the association between endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction in the Korean male population

OBJECTIVES: The aging process affects responsiveness and other functions of endothelium and vascular smooth muscle cells, predisposing the old vessels to the development of atherosclerotic lesions. Endothelial nitric oxide synthase (ecNOS) gene polymorphisms were shown to affect the occurrence of acute myocardial infarction (AMI). We hypothesized that aging may affect the association between the ecNOS gene polymorphism and AMI. METHODS: We investigated the age-related distribution of the ecNOS gene a/b polymorphism in 121 male AMI patients and 206 age-matched healthy male controls. RESULTS: The aa, ab and bb genotypes were found in 1, 49 and 156 cases among the control subjects and 5, 23 and 93 cases among the AMI patients, respectively. There was a significant correlation between the ecNOS polymorphism and AMI (p +AD0- 0.045). When the correlation was analyzed by age, the significance remained only in the group below the age of 51 (p +AD0- 0.009). The proportion of smokers was increased in the young patients when compared to the old patients (p +AD0- 0.033), indicating that smoking also has greater effect on the younger population. The incidences of hypertension and diabetes mellitus, however, were similar in both populations. CONCLUSION: Our work provides the first evidence that links ecNOS polymorphism to the risk of AMI in relation to age. Young persons who smoke or have ecNOSaa genotype may have an increased risk of developing AMI. The functional as well as structural changes associated with aging in the vascular endothelium may mask the effect of the ecNOS polymorphism in the development of AMI in old persons.

Prevalence of foam cells and helper-T cells in atherosclerotic plaques of Korean patients with carotid atheroma

BACKGROUND: Inflammation and activation of immune cells have important roles in the pathogenesis of atherosclerosis. We analyzed the involvement of various immune cells in the pathogenesis of atherosclerosis. METHODS: We investigated the presence of foam cells, lymphocytes and killer cells in 11 atherosclerotic plaque specimens removed from Korean patients who underwent carotid endoarterectomy. Atherosclerotic plaques were analyzed by immunohistochemistry using monoclonal antibody specific to foam cells (anti-CD68), pan-T cells (anti-CD3), helper-T cells (anti-CD4), cytotoxic T cells (anti-CD8), granular component of killer cells (anti-TIA-1) and pan-B cells (anti-CD20). RESULTS: Analysis revealed a general infiltration of immune cells not only in atherosclerotic plaques but also in the vascular wall adjacent to the plaque. Heavy infiltration of CD68 macrophage was observed in all cases. In addition, significant infiltration of CD3 T-lymphocytes was observed in all cases, while CD20 B-cells were observed in only a few cases. Majority of the CD3 cells was found to be CD4 helper-T cells. CD8 cytotoxic T cells and TIA-1 cells were less prominent. CONCLUSION: Analysis of the human atherosclerotic plaques suggested that helper-T cells and foam cells had a major role in the plaque development.

Expression of tumor necrosis factor receptor superfamily in carotid atheroma

BACKGROUND AND OBJECTIVES: It has been reported that various inflammatory and immune reactions are involved in the development and progression of atherosclerosis. We tried to investigate whether the TNF receptor superfamilies are involved in the development and progression of atherosclerosis. MATERIALS AND METHOD: Thirteen carotid atheroma specimens(frozen sections : 10 cases, paraffin section : 5 cases) were obtained from the patients who underwent carotid endarterectomy at Samsung Medical Center and one normal aortic tissue was obtained from a transplantation donor in brain death. In the carotid endarterectomy specimens and a normal aortic tissue , the expressions of R110(TR1), 139(TR2) and DR3(TR3), members of the TNF receptor superfamilies were evaluated by immunohistochemical staining with monoclonal antibodies. Simultaneously, we evaluated the expressions of foam cells, smooth muscle cells, T-lymphocytes and B-lymphocytes. RESULTS: Immunohistochemical analysis identified a strong expressions of foam cells and smooth muscle cells in all atheroma. But, the expression of T-lymphocytes was minimal and that of B-lymphocytes was rare. The expression of DR3(TR3) was seen in all atheroma as strongly positive. The expression of 139(TR2) was observed well in frozen sections, but not in paraffin sections. Whereas, that of R110(TR1) was observed in paraffin sections as weakly positive, but not in frozen section. The areas where the TNF receptor superfamilies were expressed correlated to the area of foam cell presence. The expression of DR3 also correlated with expression of smooth muscle cells. In normal aortic tissue, the expression of inflammatory cells or TNF receptor superfamilies was not observed except smooth muscle cells which were observed in normal artery. CONCLUSION: Foam cells and smooth muscle cells were abundantly present in atheroma. The TNF receptor superfamilies are expressed in the atheroma and the region of expression was coincident with the presence of foam cells.

Analysis of the Endothelial Nitric Oxide Synthase and -fibrinogen Gene Polymorphism in the Development of Acute Myocardial Infarction in Korean Men

BACKGROUND AND OBJECTIVE: The aging process affects the responsiveness and other functions of endothelium and vascular smooth muscle cells, predisposing the old vessels to the development of atherosclerotic lesions. Endothelial nitric oxide synthase (ecNOS) gene polymorphism was shown to affect the occurrence of acute myocardial infarction (AMI). We hypothesized that aging may affect the association between the ecNOS gene polymorphism and AMI. METHODS: We investigated the age-related distribution of the ecNOS gene a/b polymorphism in 121 male AMI patients and 206 age-matched healthy male controls. As a control, we also genotyped b-fibrinogen gene H1/H2 polymorphism in the same population. RESULTS: The aa, ab, and bb genotypes were found in 1, 49 and 156 cases among the control subjects and 5, 23 and 93 cases among the AMI patients, respectively. There was a significant association between the ecNOS polymorphism and AMI (p=0.045). When the correlation was analyzed by age, the significance remained only in the group below the age of 51 (p=0.009). The distribution of the b-fibrinogen gene H1/H2 alleles, however, was not found to be associated with development of AMI in both young (p=0.7400) and old (p=0.2160) population. CONCLUSION: Our results provide the first evidence that links ecNOS polymorphism to the risk of AMI in relation to age. Young persons who smoke or have ecNOS aa genotype may have an increased risk of developing AMI. The functional as well as structural changes associated with aging in the vascular endothelium may mask the effect of the ecNOS polymorphism in the development of AMI in old people.

Activation of monocytes, T-lymphocytes and plasma inflammatory markers in angina patients

Inflammation and activation of immune cells have important roles in the pathogenesis of atherosclerosis. We analyzed the plasma levels of inflammatory markers and the degree of activation of peripheral blood monocytes and T-lymphocytes isolated from 12 unstable angina, 12 stable angina, and 12 normal subjects. In 20%-33% of patients, monocytes expressed high basal levels of IL-8, tissue factor, IL-1beta, and monocyte chemoattractant protein-1 mRNA. Furthermore, basal mRNA levels of these cytokines showed strong correlation with each other (p < 0.01 in all combination) but not with tumor necrosis factor-alpha or transforming growth factor-beta1. Plasma level of C-reactive protein was highest in the unstable angina patients (1.63+/-0.70 mg/l) and lowest in the control subjects (0.22+/-0.08 mg/l) (P = 0.03). We also observed a high correlation between C-reactive protein level and the occurrence of minor and major coronary events during 6 months of follow-up. Activation status of T-cells, assessed by the percentage of HLA-DR positive cells, was highest in the unstable angina patients (26.8+/-1.4%) compared with that in the control (14.7+/-1.2%) (P = 0.0053). Our data represent the first case showing that the circulating monocytes in angina patients are activated to a state express numerous proatherogenic cytokines. These results may help to diagnose angina patients according to the inflammatory markers and evaluate the prognosis of the disease.