Aberrant expression of regulatory cytokine IL-35 in patients with systemic lupus erythematosus.

OBJECTIVE: This study characterizes an IL-35-mediated regulatory role in patients with systemic lupus erythematosus (SLE). METHODS: Plasma of SLE patients and healthy controls (HCs) was analyzed for the concentrations of IL-35 and soluble gp130 by using ELISA. mRNA expression of IL-35 subunit (p35 and EBI3) and its receptor (gp130 and IL-12Rß2) in peripheral blood mononuclear cells (PBMCs) was assessed by RT-qPCR. Flow cytometry was performed to evaluate the number of CD4(+)CD25(high)CD127(-)Treg cells and the expression of IL-35 receptor on the CD4+ helper (Th) cells and CD19+ B cells. Plasma collected from SLE patients and HCs was assayed for cytokine and chemokine expression by Luminex multiplex assay. RESULTS: Plasma IL-35 and soluble gp130 levels positively correlated with each other and were significantly higher in patients with severe SLE compared with HCs. Significantly higher levels of inflammatory cytokines/chemokines CCL2, CXCL8, IL-6, interferon (IFN)-γ, IL-10 and IL-17A were observed in plasma of SLE patients than HCs. mRNA levels of IL-35 and its receptor were significantly positively correlated in PBMCs from SLE patients and their levels were higher in SLE than HCs. The increase significantly correlated with changes in SLE Disease Activity Index (SLEDAI) (all p < 0.05). In addition, the number of Treg cells in severe and moderate SLE patients were both significantly lower than HCs, where the ratio of CD4(+)CD25(-)effector T cell %/CD4(+)CD25(high)CD127(-)Treg % was found to be significantly higher in severe SLE patients. Furthermore, the expression of gp130 on CD4+ Th cells and percentage of Tregs were positively correlated with each other, and both were negatively correlated with SLEDAI. CONCLUSION: Our findings indicate that high level of plasma IL-35 in active SLE patients expressed with low level of IL-35 receptor (gp130) on CD4+ Th cells. These data raise the possibility that the level of IL-35 expression in SLE patients is not sufficient to induce the production of CD4(+)CD25(high)CD127(-)Tregs, and subsequently suppress the release of inflammatory cytokines and chemokines upon inflammation.

Remission of systemic lupus erythematosus disease activity with regulatory cytokine interleukin (IL)-35 in Murphy Roths Large (MRL)/lpr mice.

The immunological mechanisms mediated by regulatory cytokine interleukin (IL)-35 are unclear in systemic lupus erythematosus (SLE). We investigated the frequency of CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) regulatory T (Treg ) and IL-10(+) regulatory B (Breg ) cells and related immunoregulatory mechanisms in a female Murphy Roths Large (MRL)/lpr mouse model of spontaneous lupus-like disease, with or without IL-35 treatment. A remission of histopathology characteristics of lupus flare and nephritis was observed in the MRL/lpr mice upon IL-35 treatment. Accordingly, IL-35 and IL-35 receptor subunits (gp130 and IL-12Rß2) and cytokines of MRL/lpr and BALB/c mice (normal controls) were measured. The increased anti-inflammatory cytokines and decreased proinflammatory cytokines were possibly associated with the restoration of Treg and Breg frequency in MRL/lpr mice with IL-35 treatment, compared to phosphate-buffered saline (PBS) treatment. mRNA expressions of Treg -related FoxP3, IL-35 subunit (p35 and EBI3) and soluble IL-35 receptor subunit (gp130 and IL12Rß2) in splenic cells were up-regulated significantly in IL-35-treated mice. Compared with the PBS treatment group, IL-35-treated MRL/lpr mice showed an up-regulation of Treg -related genes and the activation of IL-35-related intracellular Janus kinase/signal transducer and activator of transcription signal pathways, thereby indicating the immunoregulatory role of IL-35 in SLE. These in vivo findings may provide a biochemical basis for further investigation of the regulatory mechanisms of IL-35 for the treatment of autoimmune-mediated inflammation.

Autoimmunity to the p53 protein is a feature of systemic lupus erythematosus (SLE) related to anti-DNA antibodies.

The induction of anti-DNA autoantibodies in systemic lupus erythematosus (SLE) patients is problematic because mammalian DNA is poorly immunogenic at best. Here we demonstrate a chain of connected antibodies in SLE patient sera that could account for the induction of anti-DNA antibody, and possibly for some of the pathogenic features of SLE. We now report that SLE patients, in addition to anti-DNA, produce antibodies to the carboxy-terminal domain of the tumour suppressor molecule p53; this p53 domain recognizes damaged DNA. Hence, these anti-p53 antibodies could mimic damaged DNA immunologically. Indeed, SLE sera do contain anti-idiotypic antibodies to a prototypic anti-p53 antibody. Moreover, SLE anti-DNA antibodies also recognize this type of anti-p53 antibody. Indeed, binding of affinity-purified anti-DNA both to DNA and to the anti-p53 antibody could be blocked by a p53 peptide derived from the DNA-binding domain. This mimicry of the p53 DNA-binding domain by the SLE anti-DNA antibodies is functional: activation of the p53 molecule could be inhibited by such anti-DNA antibodies. Thus, anti-DNA antibodies may arise in SLE patients by a chain of idiotypic autoimmunity centered around p53 autoimmunity. The SLE anti-DNA and anti-p53 antibodies can functionally block p53 activation, and so could affect apoptosis.

TNF-alpha associated with extracellular matrix fibronectin provides a stop signal for chemotactically migrating T cells.

The migration of T cells into extravascular sites of inflammation is regulated by information derived from the molecular structure of the invaded tissue and from chemokine and cytokine gradients in the context of the extracellular matrix (ECM). Although recent studies have highlighted the role of particular chemoattractants in leukocyte migration, to date little is known about how specific combinations of contextual signals control the migration of leukocytes and their localization at sites of inflammation. Here we studied the interplay between a pleiotropic cytokine, TNF-alpha, and two prototypic chemoattractants, RANTES and stromal cell-derived factor-1alpha (SDF-1alpha), on human CD45RO+ T cells migrating within an ECM-like context. For this purpose, we used a newly constructed three-dimensional gel system designed to follow, in real time, the migration of individual leukocytes along chemotactic gradients in vitro. We found that TNF-alpha, which binds the ECM protein fibronectin and lacks adhesion- and migration-promoting effects of its own, can act as a proadhesive cytokine on T cells exposed to RANTES and SDF-1alpha. Furthermore, fibronectin-complexed TNF-alpha provided anchorage signals to the T cells as they moved directionally along chemoattractive gradients. This effect of TNF-alpha required an intact TNF-alpha receptor II subtype on the migrating T cells. The anchoring effect of TNF-alpha appears to be specific; IL-2, an integrin-activating proadhesive cytokine, does not transmit stoppage signals to T cell migration induced by RANTES. Thus, TNF-alpha present in the ECM at sites of inflammation may function to anchor T cells recruited to these sites by chemotactic signals.

Video-assisted thoracoscopic resection of an epiphrenic diverticulum with esophagomyotomy and partial fundoplication.

Videothoracoscopic stapled diverticulectomy with distal esophageal myotomy and partial fundoplasty was successfully done for a 65-year-old woman who had a large, symptomatic epiphrenic diverticulum associated with achalasia. This minimally invasive approach resulted in good symptomatic relief of dysphagia, minimal postoperative pain, a 1-day hospital stay, and early return to normal activity.

Effect of fat feeding on human intestinal apolipoprotein B mRNA levels and editing.

Our purpose was to assess the effect of a fat-rich meal on intestinal apolipoprotein B (apoB) mRNA levels and editing. We obtained jejunal biopsies from eight healthy adults in the fasting state and 3 h after a meal containing 1 g/kg of fat. In the fasting state, 93% of the apoB mRNA contained the editing sequence resulting in apoB-48 production. Feeding induced no significant changes in apoB mRNA levels or editing. Our data are consistent with the concept that the significant increase in apoB-48 within triglyceride-rich lipoproteins in postprandial plasma is not due to alterations in apoB gene expression or editing.

Effect of lovastatin on acyl-CoA: cholesterol O-acyltransferase (ACAT) activity and the basolateral-membrane secretion of newly synthesized lipids by CaCo-2 cells.

Lovastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, was used to study the regulation of cholesterol metabolism and the basolateral-membrane secretion of triacylglycerol and cholesterol in the human intestinal cell line CaCo-2. At 0.1 microgram/ml, lovastatin decreased 3H2O incorporation into cholesterol by 71%. In membranes prepared from cells incubated with lovastatin for 18 h, HMG-CoA reductase activity was induced 4-8-fold. Mevalonolactone prevented this induction. In intact cells, lovastatin (10 micrograms/ml) decreased cholesterol esterification by 50%. The reductase inhibitor decreased membrane acyl-CoA:cholesterol O-acyltransferase (ACAT) activity by 50% at 5 micrograms/ml. ACAT inhibition by lavastatin was not reversed by adding excess of cholesterol or fatty acyl-CoA to the assay. Lovastatin, in the presence or absence of mevalonolactone, decreased the basolateral secretion of newly synthesized cholesteryl esters and triacylglycerols. Lovastatin also inhibited the esterification of absorbed cholesterol and the secretion of this newly synthesized cholesteryl ester. Lovastatin is a potent inhibitor of cholesterol synthesis in CaCo-2 cells. Moreover, it is a direct inhibitor of ACAT activity, independently of its effect on HMG-CoA reductase and cholesterol synthesis.

Regulation of cholesterol metabolism in the intestine.

The small intestine is a major site of cholesterol biosynthesis and lipoprotein degradation. It is also the organ responsible for absorbing dietary and endogenously produced biliary cholesterol. Cholesterol metabolism in the intestine is regulated by factors that will alter cellular cholesterol requirements. Thus, during increased cholesterol flux, which occurs by bile acid-faciliated cholesterol absorption or by lipoprotein-mediated uptake of cholesterol, cholesterol synthetic rates decrease and esterification rates increase. The mechanisms by which dietary fats regulate intestinal cholesterol metabolism are complex. Dietary fats alter membrane fatty acid composition. Simultaneously, they also promote lipoprotein secretion and alter cholesterol absorption. Intestinal 3-hydroxyl-3-methylglutaryl coenzyme. A reductase activity is regulated by enzyme phosphorylation-dephosphorylation. The regulation of acylcoenzyme A-cholesterol acyltransferase activity by this mechanism remains controversial. Data on hormone regulation of intestinal cholesterol metabolism are not conclusive, although progesterone seems to be a potent inhibitor of acylcoenzyme A-cholesterol acyltransferase activity in intestinal cell culture and isolated cells. In a manner similar to the regulation of cholesterol metabolism in other cells, the enterocyte responds appropriately to factors that alter cholesterol flux. Therefore, changes that occur in the rates of cholesterol synthesis and esterification will reflect the cholesterol requirements of the cell.

Inhibition of acylcoenzyme A:cholesterol acyltransferase activity in CaCo-2 cells results in intracellular triglyceride accumulation.

The activity of acylcoenzyme A:cholesterol acyltransferase (ACAT) in CaCo-2 cells was inhibited by the ACAT inhibitor, 58-035. The inhibitory effect of this acylamide was specific for cholesterol esterification catalyzed by ACAT; the rates of triglyceride, phospholipid, and cholesterol synthesis were not inhibited by this agent. Cholesteryl esters were depleted in CaCo-2 cells 24 hr after inhibition of ACAT activity, whereas the unesterified cholesterol content increased by 56% after 96 hr. Moreover, inhibiting ACAT activity with 58-035 resulted in a time-dependent 2.5-fold increase in intracellular triglycerides. This accumulation of triglycerides in CaCo-2 cells was associated with a 37% increase in triglyceride synthesis by 96 hr in the presence of 58-035. Triglyceride-rich lipoprotein secretion (d less than 1.006 g/ml) was not affected by inhibiting ACAT activity for up to 6 hr. However, triglyceride-rich lipoprotein secretion was significantly decreased in CaCo-2 cells that were preincubated with 58-035 for 24 to 96 hr. Lipoproteins of density less than 1.006 g/ml that were isolated from CaCo-2 cells incubated with the ACAT inhibitor were deficient in cholesteryl esters and triglycerides compared to lipoproteins isolated from control cells. The data suggest that triglycerides accumulate in CaCo-2 cells in which ACAT activity has been inhibited by 58-035. This accumulation of triglycerides is associated with a modest increase in triglyceride synthesis and a decrease in triglyceride secretion. Altering intracellular cholesterol pools by regulating ACAT activity in the gut could result in the decrease of triglyceride transport and/or the secretion of triglyceride-rich lipoprotein particles of abnormal composition.