Andries S. Koster

Immunoglobulin-free light chains elicit immediate hypersensitivity-like responses

Immunoglobulin (Ig)-free light chains IgLC are present in serum and their production is augmented under pathological conditions such as multiple sclerosis, rheumatoid arthritis and neurological disorders. Until now, no (patho)physiological function has been ascribed to circulating Ig light chains. Here we show that IgLCs can confer mast cell–dependent hypersensitivity in mice. Antigenic stimulation results in plasma extravasation, cutaneous swelling and mast-cell degranulation. We show that IgLCs have a crucial role in development of contact sensitivity, which could be completely prevented by a novel IgLC antagonist. Although IgE and IgG1 are central to the induction of immediate hypersensitivity reactions, our results show that IgLCs have similar activity. IgLCs may therefore be a novel factor in the humoral immune response to antigen exposure. Our findings open new avenues in investigating the pathogenesis of autoimmune diseases and their treatments.

Apocynin inhibits peroxynitrite formation by murine macrophages

Peroxynitrite (ONOO−) the highly reactive coupling product of nitric oxide and superoxide, has been implicated in the pathogenesis of an increasing number of (inflammatory) diseases. At present, however, selective peroxynitrite antagonizing agents with therapeutic potential are not available. Therefore, the NADPH‐oxidase inhibitor apocynin (4‐hydroxy‐3‐methoxy‐acetophenone) was tested for its ability to inhibit peroxynitrite formation in vitro The murine macrophage cell‐line J774A.1, stimulated with IFNγ/LPS, was used as a model. Conversion of 123‐dihydrorhodamine (123‐DHR) to its oxidation product 123‐rhodamine was used to measure peroxynitrite production. Stimulated peroxynitrite formation could be completely inhibited by apocynin, by the superoxide scavenger TEMPO as well as by the nitric oxide synthase inhibitor aminoguanidine. Apocynin and aminoguanidine specifically inhibited superoxide and nitric oxide formation respectively as confirmed by measuring lucigenin enhanced chemiluminescence and nitrite accumulation. It is concluded that J774A.1 macrophages produce significant amounts of peroxynitrite, which is associated with nitric oxide production and NADPH‐oxidase dependent superoxide formation. The NADPH‐oxidase inhibitor apocynin proved to be a potent inhibitor of both superoxide and peroxynitrite formation by macrophages, which may be of future therapeutic significance in a wide range of inflammatory disorders.

Apocynin and 1400 W prevents airway hyperresponsiveness during allergic reactions in mice

The contribution of reactive nitrogen species to the development of airway hyperresponsiveness in a mouse model of allergic inflammation was investigated by the use of selective inhibitors of nitric oxide and superoxide formation. Sensitized mice, repeatedly challenged with ovalbumin showed a significant (P<0.001, n=9) increase in airway responsiveness measured using whole body plethysmography. This hyperresponsiveness was accompanied by an influx of eosinophils into the airway lumen and increased levels of ovalbumin‐specific serum IgE. Treatment of mice with the iNOS inhibitor 1400 W or the NADPH‐oxidase inhibitor apocynin did not significantly alter cellular influx into the airway lumen nor serum ovalbumin specific IgE. In contrast, apocynin as well as 1400 W inhibited ovalbumin‐induced airway hyperresponsiveness (P<0.001 and P<0.05 respectively, n=9). Furthermore, the airways of allergen challenged animals showed clear 3‐nitrotyrosine staining, which was mainly located in eosinophils. Remarkably, treatment with apocynin or 1400 W did not alter 3‐nitrotyrosine staining. These data suggest that the development of airway hyperresponsiveness during the airway inflammation upon ovalbumin challenge is dependent on the release of both superoxide and nitric oxide and is therefore likely to be dependent on reactive nitrogen species. This mechanism, however, is not reflected by 3‐nitrotyrosine formation in the airways.

Comparison of two immortalized human cell lines to study nuclear receptor-mediated CYP3A4 induction.

Since CYP3A4 is responsible for the biotransformation of over 50% of all clinically used drugs, induction results in an increased clearance of many concomitantly administered drugs, thereby decreasing treatment efficacy or, in the case of prodrugs, lead to severe intoxications. CYP3A4 induction is regulated by the pregnane X receptor, constitutive androstane receptor, and vitamin D receptor. Since these nuclear receptors show large interspecies differences, accurate prediction of nuclear receptor-mediated CYP3A4 induction in humans requires the use of human systems. Because primary cultures of human hepatocytes or enterocytes have major drawbacks like poor availability and poor reproducibility, human cell lines are a good alternative. In this study, the widely used HepG2 cell line was compared with the LS180 cell line to serve as a model to study CYP3A4 induction. There was a clear difference between the cell lines with respect to CYP3A enzyme expression and induction. In LS180, CYP3A4 was expressed and was found to be induced by prototypical nuclear receptor agonists, whereas in HepG2, CYP3A4 was nonresponsive to treatment with rifampicin, CITCO [6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-3,4-dichlorobenzyl) oxime], or calcitriol. We subsequently evaluated whether these host-cell differences also have an effect on CYP3A4 reporter gene activity. We clearly show that there are differences in CYP3A4 reporter activity between the cell lines, and based on these results and those found on mRNA and protein level, we conclude that LS180 is a more suitable cell line to study CYP3A4 induction than the widely used HepG2.

Depletion of ATP but not of GSH affects viability of rat hepatocytes

The purpose of this study was to examine the role of glutathione depletion and alterations in the energy status in the induction of acute cytotoxicity to freshly isolated rat hepatocytes. Depletion of intracellular glutathione by diethyl maleate and phorone to levels below 5% of control did not induce loss of viability nor loss of intracellular ATP. Ethacrynic acid, a compound known to deplete mitochondrial GSH in addition to cytosolic GSH, induced cell killing after a depletion of ATP, next to GSH depletion. The results confirmed that depletion of intracellular glutathione alone does not necessarily result in cell killing. Only when glutathione depletion is succeeded by reduction in ATP levels, loss of cell viability is observed. The relationship between alterations in the energy status and the induction of cell death was further substantiated by inhibition of glycolytic and mitochondrial ATP generation. Treatment of hepatocytes either with iodoacetic acid to inhibit glycolysis (in hepatocytes from fed rats) or with potassium cyanide to inhibit mitochondrial respiration (in hepatocytes from both fed and fasted rats) revealed that depletion of intracellular ATP could lead to lethal cell injury. The susceptibility of cells to metabolic inhibition was better reflected by the rate of reduction in the energy charge than by the reduction of ATP alone. In conclusion, our results suggest that alterations of the energy status may be a critical event in the induction of irreversible cell injury. Depletion of cellular GSH is only cytotoxic when followed by a reduction of the energy charge.

Delayed-type hypersensitivity-induced increase in vascular permeability in the mouse small intestine: inhibition by depletion of sensory neuropeptides and NK1 receptor blockade.

1 This study investigates the effects of capsaicin‐induced depletion of sensory neuropeptides and of neurokinin! (NK1) receptor blockade on delayed‐type hypersensitivity (DTH)‐induced changes of vascular permeability in the small intestine of the mouse.

Critical Role for Mast Cells in the Pathogenesis of 2,4-Dinitrobenzene-Induced Murine Colonic Hypersensitivity Reaction

The immunological mechanisms underlying the role of mast cells in the pathogenesis of inflammatory bowel disease (IBD) are poorly defined. In this study, non-IgE mediated colonic hypersensitivity responses in BALB/c mice induced by skin sensitization with dinitrofluorobenzene (DNFB) followed by an intrarectal challenge with dinitrobenzene sulfonic acid featured as a model to study the role of mast cells in the development of IBD. Vehicle- or DNFB-sensitized mice were monitored for clinical symptoms and inflammation 72 h after dinitrobenzene sulfonic acid challenge. DNFB-sensitized mice developed diarrheic stool, increased colonic vascular permeability, hypertrophy of colonic lymphoid follicles (colonic patches), and showed cellular infiltration at the microscopic level. Increased numbers of mast cells were found in the colon of DNFB-sensitized mice located in and around colonic patches associated with elevated levels of mouse mast cell protease-1 in plasma indicating mast cell activation. Colonic patches of DNFB mice, stimulated in vitro with stem cell factor indicated that an increase in TNF-α levels in the colon is mainly mast cell originated. Finally, neutrophil infiltration was observed in the colon of DNFB-sensitized mice. Induction of this model in mast cell-deficient WBB6F1 W/Wv mice shows a profound reduction of characteristics of the colonic hypersensitivity reaction. Reconstitution with bone marrow-derived mast cells in WBB6F1 W/Wv mice fully restored the inflammatory response. This study demonstrates the importance of mast cells in the development of clinical symptoms and inflammation in the presented murine model for IBD.

Glucuronidation in the rat intestinal wall: Comparison of isolated mucosal cells, latent microsomes and activated microsomes

Glucuronidation and sulphation of 1-naphthol and 7-hydroxycoumarin was studied in isolated rat intestinal epithelial cells and in microsomes prepared from these cells. In the isolated cells formation of 1-naphthol sulphate could not be detected. Sulphate conjugates of 7-hydroxycoumarin constitute a minor portion of total conjugates formed. Maximum glucuronidation rates for 1-naphthol and 7-hydroxycoumarin do not differ significantly from each other (approximately 12.5 nmoles/min X g intestine). The intestinal microsomal UDP-glucuronosyltransferase, prepared from isolated cells, could be activated in vitro by Triton X-100 and MgCl2. Activation increased both Kappm and Vmax for 1-naphthol; Kappm for UDP-glucuronic acid was decreased by activation with MgCl2 but increased again by further addition of Triton X-100. In fully activated microsomes Kappm for 1 naphthol was 69.7 +/- 13.9 microM and Vmax was 70.0 +/- 3.9 nmoles/min X mg microsomal protein; Kappm for UDP-glucuronic acid was 0.67 +/- 0.06 mM. The glucuronidation rate (expressed as nmoles/min X g intestine) in microsomes is substantially higher than in isolated cells. It appears that glucuronidation in intact cells is limited by factors other than the extracellular substrate concn. Both cellular uptake of the substrate and availability of UDP-glucuronic acid can play a significant role. It is concluded that isolated mucosal cells are more suitable for predicting intestinal first-pass metabolism of phenolic xenobiotics than intestinal microsomes, because cellular substrate uptake and cosubstrate availability appear to be important determinants of the maximum glucuronidation rate.

Alterations in energy status by menadione metabolism in hepatocytes isolated from fasted and fed rats

The biochemical mechanism of cytotoxicity, induced by the quinoid compound 2-methyl 1,4-naphthoquinone (menadione), was investigated in hepatocytes freshly isolated from fasted and fed rats. Hepatocytes from fasted rats were significantly more vulnerable to the toxicity of menadione than hepatocytes from fed rats. Menadione (150 microM) induced a 50% loss of viability of cells (LT50) from fasted rats after 55 min of incubation, whereas a LT50 of 80 min was observed after exposure of hepatocytes from fed rats to menadione. Glutathione and NADPH levels were rapidly depleted by menadione metabolism. This depletion was sustained during the incubation period. No significant differences were found in the time course and extent of the menadione-induced glutathione and NADPH depletion in hepatocytes of both nutritional states. Menadione also affected the energy status of the hepatocytes. The ATP content of cells from fasted rats decreased to 50% (AT50) within 18 min of exposure to menadione, whereas a 50% loss of ATP content of hepatocytes from fed rats was reached at 65 min. In contrast to depletion of glutathione and NADPH, the time course and extent of menadione-induced ATP depletion correlated well with the time of onset and rate of cell killing. Our results suggest that menadione metabolism may interfere with both mitochondrial and glycolytic ATP production. Depletion of ATP might be a critical step in menadione-induced cytotoxicity.

Ig-Free Light Chains Play a Crucial Role in Murine Mast Cell-Dependent Colitis and Are Associated with Human Inflammatory Bowel Diseases

Traditionally, mast cells were regarded as key cells orchestrating type I hypersensitivity responses. However, it is now recognized that mast cells are widely involved in nonallergic (non-IgE) chronic diseases. Also, in inflammatory bowel disease (IBD), a disease not associated with increased IgE concentrations, clear signs of activation of mast cells have been found. In this study, we investigated if Ig-free L chain-induced hypersensitivity-like responses through activation of mast cells could contribute to the pathophysiology of IBD. As a mast cell-dependent model for IBD, mice were skin-sensitized with dinitrofluorobenzene followed by intrarectal application of the hapten. In this murine IBD model, F991 prevented mast cell activation and also abrogated the development of diarrhea, cellular infiltration, and colonic lymphoid follicle hyperplasia. Furthermore, passive immunization with Ag-specific Ig-free L chains (IgLCs) and subsequent rectal hapten challenge elicited local mast cell activation and increased vascular permeability in the colon of mice. Clinical support is provided by the observation that serum concentrations of IgLCs of patients suffering from Crohn’s disease are greatly increased. Moreover, increased presence of IgLCs was evident in tissue specimens from colon and ileum tissue of patients with IBD. Our data suggest that IgLCs may play a role in the pathogenesis of IBD, which provides novel therapeutic means to prevent or ameliorate the adverse gastrointestinal manifestations of IBD.