Chitotriosidase gene expression in Kupffer cells from patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Non-alcoholic steatohepatitis (NASH) is a clinicopathological condition characterised by a necroinflammatory disorder with fatty infiltration of the hepatocytes. The molecular mechanisms involved in the anomalous behaviour of liver cells have only partially been determined. Human chitotriosidase (Chit) is a chitinolytic enzyme mainly produced by activated macrophages. The aim of this study was to investigate the expression of the chitinase-like gene in Kupffer cells, to determine how chitotriosidase may be implicated in the progression from uncomplicated steatosis to steatohepatitis with progressive fibrosis. METHODS: 75 subjects were studied: 40 with NASH, 20 with simple steatosis, and 15 normal controls. Kupffer cells obtained from liver biopsies were used to detect CHIT expression, superoxide anion (O2-), lipid peroxidation, and tumour necrosis factor alpha (TNFalpha) and ferritin levels. RESULTS: CHIT expression differed markedly in livers from normal controls and in those from patients with simple steatosis or non-alcoholic steatohepatitis. A significant correlation between mRNA CHIT and O2-, lipid peroxidation, TNFalpha, and ferritin levels was observed in both NASH and simple steatosis. CONCLUSIONS: Human Kupffer cells in NASH patients overproduce chitotriosidase. At the highest levels of production, this enzyme may play a role in increasing the risk for a poor outcome in steatohepatitis.

Plasma membrane localization of palmitoylated tubulin.

PC12 pheochromocytoma cells incorporate [(3)H]palmitic acid into tubulin in a time- and cell-density-dependent manner. The plasma membrane-enriched fraction contains most of the radioactivity of the membrane pellet. While palmitoylated tubulin is found in both the cytoplasm and particulate fraction, the bulk of [(3)H]palmitic acid bound to tubulin is present in the crude membrane pellet and the tubulin extracted from the plasma membrane is more heavily palmitoylated than that extracted from endoplasmic reticulum. Detergent-extracted tubulin from plasma membrane is, to a large extent, polymerization competent; a substantial fraction, increasing as a function of labeling time, is not hydroxylamine-labile. The requirement for detergent extraction, the accompanying changes in tubulin properties and the present findings of preferential incorporation of labeled tubulin into plasma membranes, make it clear that direct incorporation of tubulin into the plasma membrane can occur.

Autopalmitoylation of tubulin.

Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2-3 h at 36-37 degrees C under native conditions. Both alpha and beta tubulin are labeled, and 63-73% of the label was hydroxylamine-labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of approximately 13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylation sites. Palmitoylation increases the electrophoretic mobility of a portion of alpha tubulin toward the beta band. Palmitoylated tubulin binds a colchicine analogue normally, but during three warm/cold polymerization/depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA.

Palmitoylation of tubulin.

Tubulin is a very water soluble protein, yet a significant portion is firmly associated with cell membranes. Because recent work has shown that palmitoylation is a dynamic process that can alter the targeting of proteins to membranes, we tested whether or not tubulin could be palmitoylated to account for its membrane location. Tubulin acylation was measured by incorporation of [3H]palmitate into PC12 cells in culture. We found palmitoylated tubulin in both cell pellet and cytosol with a higher concentration in the former. EGF-stimulated PC12 cells incorporated the same amount of palmitate per unit protein but the proportion in the membrane fraction was enhanced. More palmitate of the pellet was found in alpha than beta tubulin; EGF stimulation primarily increased palmitate in beta tubulin. In addition we found that palmitic acid was present both as thioesters and as oxyesters. We suggest that palmitoylation may contribute to the membrane localization of tubulin and can be regulated by growth factors.

Glucose may induce cell death through a free radical-mediated mechanism.

It has been reported that glucose may autooxidize generating free radicals which have been hypothesized to induce important cellular abnormalities. To investigate the cell damage induced by glucose-dependent oxidative stress, the FRTL5 cell strain was incubated in 10 or 20 mM glucose, either alone or in the presence of buthionine-sulfoximine, a transition state inhibitor that blocks glutathione synthesis. We found indeed that buthionine-sulfoximine greatly inhibited glutathione production and increased malondialdehyde (a marker of oxidative cell damage) levels, especially in 20mM glucose. We also found that, when glutathione production was inhibited, 10mM glucose induced apoptosis and 20 mM glucose induced necrosis. These data show that the glucose-dependent cell damage is a function of glutathione production. They also show that such glucose-dependent free radical production may be critical for determining cell damage, even for small variations as the ones we tested (from 10 to 20 mM glucose).

Markers of T lymphocyte activation in HLA-B8, DR3 positive individuals.

Many autoimmune diseases are associated in Caucasians with HLA-B8 and/or HLA-DR3 antigens. There is evidence that bearers of these antigens may display significant changes in immune parameters when compared to individuals not having these antigens. Recently, increased numbers of blood activated T lymphocytes have been reported in the majority of these diseases. The increase in activated blood T lymphocytes is paradoxically characterized by an in vitro impairment of T cell activation. Particularly, an inadequate production of interleukins has been observed. We have studied blood levels of activated T cells in HLA-typed, healthy subjects. The results show that the percentage of activated T cells, as recognized by monoclonal antibodies anti-CD25, anti-Ia and anti-MLR3, was more frequent in HLA-B8, DR3 positive individuals. On the other hand, in the 24 h, PHA stimulated cultures IL-2, IFN-gamma and the percentage of T cells CD25 positive were decreased. Thus, there was an apparent discrepancy between the increase of blood activated T cells and the in vitro impaired T cell activation. Since there is evidence that HLA-B8, DR3 positive subjects are genetically low responders, a possible reason for the discrepancy might be their relative inability to remove antigenic stimuli from the body. In this case, the increased number of activated blood T cells may reflect a cellular activation caused by persistent antigenic stimulation.

Interleukin 2 and soluble interleukin 2-receptor secretion defect in vitro in newly diagnosed type I diabetic patients.

In this study, we investigated whether an interleukin 2 (IL-2) secretion defect by peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with phytohemagglutinin (PHA-M) occurs in either newly diagnosed or long-standing type I (insulin-dependent) diabetic patients and whether it is accompanied by a dysregulation of soluble IL-2-receptor (IL-2RS) production. PBMC cultures (2.5 x 10(6) cells), unstimulated or stimulated with PHA-M (25 micrograms/ml), from 20 type I diabetic patients (10 with time since onset less than 3 mo and 10 with long-term diabetes of less than 3 yr) and 10 control subjects were studied for the production of IL-2 and IL-2RS in their respective supernatants. No difference was found in IL-2 production in unstimulated cultures of type I patients compared with control subjects, although a significant decrease from PHA-M-stimulated cultures were seen (newly diagnosed, 1.7 +/- 0.3 ng/2.5 x 10(6) cells; long-standing, 2.2 +/- 0.3 ng/2.5 x 10(6) cells; P less than .001 and P less than .05, respectively) compared with control subjects (3.6 +/- 0.4 ng/2.5 x 10(6) cells). In regard to the production of IL-2RS, no difference exists for unstimulated cultures, whereas, after PHA-M stimulation, both newly diagnosed and long-term-diabetic patients showed a decrease in the IL-2RS levels (318 +/- 50 and 331 +/- 62 U/2.5 x 10(6) cells; P less than .02 and P less than .05, respectively) compared with normal subjects (463 +/- 34.2 U/2.5 x 10(6) cells). Thymus-activated cell phenotypes confirmed the T-lymphocyte activation after a 48-h culture period.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociated production of interleukin-2 and immune gamma-interferon by phytohaemoagglutinin stimulated peripheral mononuclear cells in type 1 (insulin-dependent) diabetes.

In order to analyze the first steps of T cell activation in type 1 diabetes we studied in vitro IL-2 and gamma-IFN production by peripheral blood mononuclear cells after 24 h PHA stimulation. There was a significant decrease in IL-2 production by mononuclear cells of the diabetic patients with respect to the controls. No significant difference was observed between the diabetic patients and the healthy subjects as regards gamma-IFN production. These observations may be interesting in relation to the pathogenetic mechanisms involved in type 1 diabetes. In particular, normal gamma-IFN production may indicate integrity of the natural killer circuit.