The role of Kupffer cells in carbon tetrachloride intoxication in mice.

Carbon tetrachloride (CCl(4))-induced acute hepatitis is assumed to involve two phases. The initial phase, initiated within 2 h after CCl(4) administration, involves the generation of reactive oxygen species. The second phase is assumed to start about 8 h subsequent to CCl(4) administration and involves the oxidant-induced activation of Kupffer cells, which release various pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). We investigated the role of Kupffer cells during CCl(4) intoxication using Nucling-knockout mice (the KO group), in which the number of Kupffer cells is largely reduced. Plasma alanine transaminase and aspartate transaminase levels demonstrated that the liver necrosis during the second phase was significantly alleviated in the KO group compared with that in the wild-type mice (the WT group). Plasma TNF-α concentrations in the WT group significantly increased 24 h after CCl(4) intoxication, whereas those in the KO group did not significantly increase. Plasma IL-6 levels also significantly increased in the WT group 24 h after CCl(4) administration, but those in the KO group did not increase at any time point. These results indicated that excess reactions of Kupffer cells, once primed by oxidants, were involved in the exacerbation of oxidative stress and liver damage during the second phase of CCl(4) intoxication.

Oxidative stress in the ischemic and non-ischemic parts of the rat liver after two-thirds ischemia/reperfusion.

Rat liver was subjected to two-thirds warm ischemia for 45 min and reperfusion (I/R) to evaluate the resulting oxidative stress. The plasma alanine aminotransferase and aspartate aminotransferase activities were significantly higher than those in the sham group 1.5-24 h after I/R, showing extensive liver cell death. The level of oxidative stress was compared between the ischemic and non-ischemic regions based on the change in antioxidative vitamins C and E. The vitamin C level was significantly decreased during I/R in both the ischemic and non-ischemic regions 0, 1.5, 3, 6, 12, and 24 h after the start of reperfusion, showing enhanced oxidative stress even in the non-ischemic lobules. This decrease of vitamin C in the ischemic region was significantly higher than that in the non-ischemic lobules, while the vitamin E content was decreased only in the ischemic lobes, demonstrating higher oxidative stress in the ischemic region than that in the non-ischemic region. Early transient activation of cytoprotective extracellular signal-related kinase (ERK) was apparent in both the ischemic and non-ischemic lobules, reflecting oxidative stress in both regions. Early transient activation of c-Jun NH(2)-terminal kinase (JNK) was only apparent in the ischemic region, corresponding to extensive oxidative stress and liver cell death. These results demonstrate that significant oxidative stress was induced, but that JNK leading to cell death was not activated in the non-ischemic part of the liver.

Increase of MZB1 in B cells in systemic lupus erythematosus: proteomic analysis of biopsied lymph nodes.

BACKGROUND: Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which dysregulation of B cells has been recognized. Here, we searched for potential biomarkers of SLE using liquid chromatography-tandem mass spectrometry (LC-MS). METHODS: Lymph nodes from SLE patients and controls were analyzed by LC-MS. To validate the identified molecules, immunoblotting and immunohistochemistry were performed and B cells from SLE patients were analyzed by quantitative RT-PCR. B-cell subsets from NZB/W F1 mice, which exhibit autoimmune disease resembling human SLE, were analyzed by flow cytometry. Endoplasmic reticulum (ER) stress was induced by tunicamycin and the serum concentration of anti-dsDNA antibodies was determined by ELISA. TUNEL methods and immunoblotting were used to assess the effect of tunicamycin. RESULTS: MZB1, which comprises part of a B-cell-specific ER chaperone complex and is a key player in antibody secretion, was one of the differentially expressed proteins identified by LC-MS and confirmed by immunoblotting. Immunohistochemically, larger numbers of MZB1+ cells were located mainly in interfollicular areas and scattered in germinal centers in specimens from SLE patients compared with those from controls. MZB1 colocalized with CD138+ plasma cells and IRTA1+ marginal zone B cells. MZB1 mRNA was increased by 2.1-fold in B cells of SLE patients with active disease (SLE Disease Activity Index 2000 ≥ 6) compared with controls. In aged NZB/W F1 mice, splenic marginal zone B cells and plasma cells showed elevated MZB1 levels. Tunicamycin induced apoptosis of MZB1+ cells in target organs, resulting in decreased serum anti-dsDNA antibody levels. Additionally, MZB1+ cells were increased in synovial tissue specimens from patients with rheumatoid arthritis. CONCLUSIONS: MZB1 may be a potential therapeutic target in excessive antibody-secreting cells in SLE.

Effect of dietary cholesterol and high fat on ceramide concentration in rat tissues.

OBJECTIVE: Recent studies have indicated that plasma sphingomyelin levels and sphingomyelinase activity are risk factors for atherosclerosis. Therefore, it is suggested that ceramides, which are hydrolyzed products of sphingomyelin and a biologically active lipid causing apoptosis in a variety of cells, have an important role in the incidence of atherosclerosis. In this study, we examined whether cholesterol- and fat-enriched diets, which are causes of atherosclerosis, affect ceramide metabolism. In addition, we found a relation among lipid markers of atherosclerosis such as cholesterol, triacylglycerol, and ceramide concentrations. METHODS: Male Wistar rats were fed a diet supplemented with 1% cholesterol or 30% high-fat diet for 8 wk. Tissue ceramide levels were analyzed using electrospray tandem mass spectrometry. RESULTS: The major ceramides in plasma and the liver were C24:0 and C24:1. The major ceramides in adipose tissues were C16:0 and C24:0. Therefore, the ceramide composition of the adipose tissues was different from that of plasma and the liver. In addition, total ceramide levels in plasma and the adipose tissues of rats fed cholesterol were higher than those in the control group. CONCLUSION: The accumulation of cholesterol caused an increase in ceramides, which might be a new risk factor for atherosclerosis.

Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients.

Transgelin-2 (TAGLN2) is an actin-binding protein that controls actin stability and promotes T cell activation. TAGLN2 is also expressed on B-cells but its function in B-cells is unknown. We found that TAGLN2-expressing B-cells were localized in the germinal center (GC) of secondary lymphoid tissues and TAGLN2 mRNA was significantly upregulated after IgM+IgG stimulation in primary human B-cells, suggesting that TAGLN2 was upregulated upon B-cell activation. In support of this, lymph nodes (LNs) from patients with systemic lupus erythematosus (SLE), in which the intense GC activity have been recognized, showed increased TAGLN2 expression in B-cells compared to control LNs. Moreover, TAGLN2+B-cells were distributed widely not only in the GC but also in the perifollicular areas in SLE LNs. In contrast, CD19+ B-cells and CD19+CD27+ memory-B cells in peripheral blood of SLE patients showed no increase in TAGLN2 mRNA. Two-photon excitation microscopy of Raji cells demonstrated that TAGLN2 colocalized with F-actin and moved together to the periphery upon stimulation. TAGLN2-knockdown in Raji cells resulted in impaired phosphorylation of PLCγ2 leading to inhibition of cell migration. Microarray analysis of TAGLN2-knockdown Raji cells showed decreased expression of the genes associated with immune function including CCR6 and as well as of those associated with regulation of the actin cytoskeleton including ABI2, compared to controls. These results suggest that TAGLN2 might regulate activation and migration of B-cells, in particular, the entry of activated B-cells into the follicle. We also suggest that TAGLN2 could be used as a marker for activated B-cells.

High Expression of Galectin-3 in Patients with IgG4-Related Disease: A Proteomic Approach.

OBJECTIVES: Immunoglobulin G4-related disease (IgG4-RD) is a multiorgan condition manifesting itself in different forms. This study aimed to investigate protein expression profiles and to find the possible biomarker for IgG4-RD by liquid chromatography mass spectrometry (LC-MS) using tissue sections in IgG4-RD patients. METHODS: Protein expression profiles in five IgG4-related pancreatitis and three normal pancreatic samples were compared using LC-MS and were validated by quantitative real-time PCR (qRT-PCR), immunoblotting, and immunohistochemistry. ELISA was employed in the serum of 20 patients with systemic IgG4-RD before and during steroid treatment. RESULTS: LC-MS indicated that the levels of 17 proteins were significantly higher and 12 others were significantly lower in IgG4-related pancreatitis patients compared to controls. Among these proteins, galectin-3 levels were 13-fold higher in IgG4-related pancreatitis (P < 0.01). These results were confirmed by immunoblotting and qRT-PCR. The average number of galectin-3 + cells in various organs of IgG4-RD patients, including salivary glands, lungs, and lymph nodes, was higher than in controls. Galectin-3 was detectable in macrophages, dendritic cells, and stromal myofibroblast-like cells, but not in lymphocytes by immunofluorescence staining. Serum galectin-3 levels were higher in patients with IgG4-RD compared with healthy donors and remained high during steroid therapy. CONCLUSION: Galectin-3 was overexpressed in IgG4-RD and the levels were indirectly related to clinical activity.

Nanoscale Mechanical Stimulation Method for Quantifying C. elegans Mechanosensory Behavior and Memory.

Withdrawal escape response of C. elegans to nonlocalized vibration is a useful behavioral paradigm to examine mechanisms underlying mechanosensory behavior and its memory-dependent change. However, there are very few methods for investigating the degree of vibration frequency, amplitude and duration needed to induce behavior and memory. Here, we establish a new system to quantify C. elegans mechanosensory behavior and memory using a piezoelectric sheet speaker. In the system, we can flexibly change the vibration properties at a nanoscale displacement level and quantify behavioral responses under each vibration property. This system is an economic setup and easily replicated in other laboratories. By using the system, we clearly detected withdrawal escape responses and confirmed habituation memory. This system will facilitate the understanding of physiological aspects of C. elegans mechanosensory behavior in the future.