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.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

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

Abstract 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

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

<div><p>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 <i>TAGLN2</i> 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. <i>TAGLN2</i>-knockdown in Raji cells resulted in impaired phosphorylation of PLCγ2 leading to inhibition of cell migration. Microarray analysis of <i>TAGLN2</i>-knockdown Raji cells showed decreased expression of the genes associated with immune function including <i>CCR6</i> and as well as of those associated with regulation of the actin cytoskeleton including <i>ABI2</i>, 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.</p></div

A larger number of TAGLN2⁺ B-cells is observed in lymph nodes and kidneys in SLE patients compared to those in controls.

<p>(A-B) Double immunofluorescence analysis of TAGLN2 and the B-cell marker CD20 is shown. Nuclei were stained with DAPI. (A) In control lymph nodes, TAGLN2⁺ B-cells were observed in follicular areas (original magnification, x200). (B) TAGLN2⁺B-cells were distributed from the follicular/germinal center (GC) to perifollicular areas in SLE lymphadenopathy (original magnification, x200). HE staining on the left (original magnification, x40). (C) The ratio of TAGLN2+ cells to CD20+ cells in SLE and controls was calculated. Significantly higher numbers of TAGLN2-positive cells were seen in B-cells in lymph nodes associated with SLE (n = 5) compared to those in controls (n = 5). Data are presented as the mean ± SEM. *p<0.05. (D) Right, a few TAGLN2⁺ CD20⁺ B-cells (arrows) are seen within B-cell aggregates in the interstitium of lupus nephritis, whereas no TAGLN2⁺ B-cells are observed in the control kidney sample (Left). (original magnification, x400). (E) Left, <i>TAGLN2</i> mRNA levels in CD19⁺ B-cells in healthy donors (HC, n = 6) and SLE patients (n = 9) as determined by qRT-PCR (mean ± SEM, 0.98 ± 0.10 in HC and 0.79 ± 0.04 in SLE) (p>0.05). Right, <i>TAGLN2</i> mRNA levels in CD19⁺CD27⁺ memory B-cells in healthy donors (HC, n = 11) and SLE patients (n = 14) (the mean of SEM, 1.0 ± 0.11 in HC and 1.2 ± 0.14 in SLE) (p>0.05.).</p

TAGLN2 colocalizes with F-actin and moves together to the periphery after stimulation. TAGLN2-GFP overlaps with LifeAct-RFP in Raji B cells at the outer actin ring.

<p>Raji cells co-transfected with TAGLN2-GFP and the actin probe LifeAct-RFP were stimulated with IgM+IgG, and the localization of each protein was then followed over the next 18 min using time lapse fluorescence microscopy. Top: TAGLN2-GFP (Green), middle: LifeAct-RFP (Magenta), bottom: merged image. The images marked as 0 were acquired before stimulation. Both the actin and TAGLN2 signals increased in intensity and thickness after IgM+IgG stimulation and were depleted from the central area of the cells and moved together to the periphery.</p

Top 30 genes downregulated in TAGLN2 knockdown versus control cells.

<p>Top 30 genes downregulated in TAGLN2 knockdown versus control cells.</p

<i>TAGLN2</i> knockdown results in impaired phosphorylation of PLCγ, which is related to the actin-linked signaling pathway and inhibition of cell migration.

<p>(A) The total protein levels of PLCγ2, PI3K, ERK, and Akt and the levels of their phosphorylated forms after 10 min IgM+IgG stimulation of <i>TAGLN2</i>-knockdown (KD) Raji cells transfected with TAGLN2 siRNA, or of control cells with scrambled siRNA (Scramble), were detected by immunoblotting. Actin was blotted as a loading control. Suppression of TAGLN2 protein expression and PLCγ2 phosphorylation was seen in the <i>TAGLN2</i>-KD Raji cells compared with the scrambled siRNA transfected cells. (B) Normalized expression levels of each protein and each phosphorylated protein assessed using membrane densitometry. Three independent experiments were performed. *p<0.05. (C) Significant inhibition of CXCL13-dependent <i>TAGLN2</i> KD Raji cell chemotaxis as compared with negative controls. *p<0.05. RFU, relative fluorescence units. Migratory cells were lysed and quantified using fluorescent dye. The relative quantification was used to determine the change between various samples. Data were normalized by designating one sample in negative controls as equal to 1. Then, the ratiometric results were used to scale all values relative to that sample. (D) The CXCR5 and IgM expression levels were assessed by flow cytometry in <i>TAGLN2</i> KD and control Raji cells. <i>TAGLN2</i> KD appeared to have no effect on the surface expression of CXCR5 and IgM. Gray shading indicates isotype control.</p

Down-regulated genes related to the immune system.

<p>Down-regulated genes related to the immune system.</p