Anouk Waeytens

Caspase inhibition causes hyperacute tumor necrosis factor-induced shock via oxidative stress and phospholipase A2

Dysregulated apoptotic cell death contributes to many pathological conditions, including sepsis, prompting the suggestion that caspase inhibition to block apoptosis could have useful therapeutic applications. Because the cytokine tumor necrosis factor (TNF, also known as TNF-α) is both pro-apoptotic and pro-inflammatory and is involved in septic shock, we tested whether caspase inhibition would alleviate TNF-induced toxicity in vivo. General caspase inhibition by the protease inhibitor zVAD-fmk exacerbated TNF toxicity by enhancing oxidative stress and mitochondrial damage, resulting in hyperacute hemodynamic collapse, kidney failure and death. Thus, survival of TNF toxicity depends on caspase-dependent processes. Our results demonstrated the pathophysiological relevance of caspase-independent, ROS-mediated pathways in response to lethal TNF-induced shock in mice. In addition, survival of TNF toxicity seemed to require a caspase-dependent protective feedback on excessive reactive oxygen species (ROS) formation and phospholipase A2 activation.

The role of heregulin-α as a motility factor and amphiregulin as a growth factor in wound healing

Wound healing is a complex process of which growth and motility are essential features. The aim of this study was to search for keratinocyte‐derived secreted factors that may play a role in these mechanisms, and their corresponding receptors. Growth and motility factors were purified from conditioned medium from cultured primary keratinocytes. Receptor and growth factor expression profiles were investigated by immunohistochemical, western blotting, and in situ hybridization analysis on cultured keratinocytes and tissue sections derived from chronic wounds. The most potent autocrine growth factor for keratinocytes, which it was possible to purify and sequence from keratinocyte‐conditioned medium, is amphiregulin. Its receptor HER‐1 is up‐regulated on the membranes of keratinocytes lining the edge of the wound. From the same keratinocyte‐conditioned medium, heregulin‐α was purified as a potent motility factor for keratinocytes. Its receptor is HER‐3, which is up‐regulated on the membranes of keratinocytes lining the edge of the wound and on keratinocytes that had migrated towards the centre of the wound. HER‐4 — another receptor for heregulin‐α — is weakly present in occasional cells near the edge of the wound. The co‐receptor for HER‐3 and HER‐4 is HER‐2/neu, which is also present in epidermal cells but not overexpressed. This study shows that heregulin‐α is a potent motility factor for normal epithelial cells and that it plays a central role in the process of wound healing of stratified epithelia. Heregulin‐α has already been shown to be the motility factor leading to migration of HER‐2/neu‐overexpressing breast cancer cells. The role of amphiregulin as a growth factor and of heregulin‐α as a motility factor for keratinocytes in epidermal and mucosal wound healing parallels their motility and growth induction in carcinogenesis. Copyright

Murine M cells express annexin V specifically.

The specialized epithelium covering the lymphoid follicles of Peyers patches in the gut mediates transcytosis of antigens to the underlying immune cells, mainly through the membranous, or M, cells. At present, the molecular processes involved in the mucosal immune response, and in antigen transport across the follicle‐associated epithelium (FAE) and M cells, are poorly understood. To characterize FAE and M cells, we compared the gene expression profiles of small intestine FAE and villus epithelium (VE) in BALB/c mice by microarray analysis; 91 genes were found to be up‐regulated and four down‐regulated at least two‐fold (p < 0.01) in the FAE. The differential expression of a subset of these genes was shown to be confirmed by quantitative RT‐PCR. Using immunohistochemistry on BALB/c Peyers patches, cathepsin H and clusterin expression was increased in the FAE compared to the VE. Moreover, we demonstrated M cell‐specific expression of annexin V, which has recently been reported to be important in endocytic transport and membrane scaffolding, suggesting that annexin V has a function in M cell‐mediated transcytosis. Copyright

Human mast cells express leptin and leptin receptors

Mast cells are immune cells that produce and secrete a variety of mediators and cytokines that influence various inflammatory and immune processes. Leptin is a cytokine regulating metabolic, endocrine as well as immune functions via the leptin receptor which is expressed by many immune cells. However, there are no data about leptin receptor expression in mast cells. Immunohistochemical and immunofluorescent double stainings showed the expression of leptin and leptin receptors in mast cells in human skin and several parts of the respiratory, gastrointestinal and urogenital tract. Leptin was expressed in mast cells expressing the classification marker chymase, whereas a variable expression was observed in tryptase positive mast cells. For leptin receptors, the expression pattern was tissue dependent and not related to tryptase or chymase expression. Our results demonstrate the expression of leptin and leptin receptors on mast cells, suggesting paracrine and/or autocrine immunomodulatory effects of leptin on mast cells.

Inflamed intestinal mucosa features a specific epithelial expression pattern of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first step in the degradation of tryptophan, an essential amino acid. During inflammation IDO can be induced in different cell types resulting in local tryptophan depletion. This inhibits T cell proliferation and may induce apoptosis. High expression of IDO was previously found in inflammatory bowel disease and is thought to represent a mechanism for downregulation of the local immune response. Our aim is to investigate the expression pattern of IDO in normal and inflamed murine and human intestinal mucosa. Immunohistochemical staining for IDO was performed on paraffin sections of colon of two mouse models for colitis and their controls and on paraffin sections of human ileum and colon in normal and two different inflammatory conditions, namely inflammatory bowel disease and diverticulitis. IDO immunohistochemistry showed similar results in murine and human tissue. In normal, as well as in inflamed mucosa, some mononuclear cells, fibroblasts and endothelial cells were positive for IDO. In inflamed mucosa a specific expression pattern of epithelial IDO was found where epithelial cells flanking ulcers or bordering crypt abscesses showed high IDO expression. Moreover, in human intestinal inflammation, IDO was expressed in ulcer associated cell lineage. Since bacterial invasion is more pronounced in erosions and in crypt abscesses and since IDO activity and the resulting local tryptophan depletion can cause growth arrest of several tryptophan-dependent microorganisms, IDO expression in the vicinity of interruptions of the epithelial barrier may point to a role for IDO as a local anti-infectious agent. Furthermore, expression of IDO at the margin of ulcerations and in the reparative ulcer-associated cell lineage suggests involvement of IDO in repair processes.

Evidence for a Potential Role of Metallothioneins in Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBDs) are a group of chronic, relapsing, immune-mediated disorders of the intestine, including Crohns disease and ulcerative colitis. Recent studies underscore the importance of the damaged epithelial barrier and the dysregulated innate immune system in their pathogenesis. Metallothioneins (MTs) are a family of small proteins with a high and conserved cysteine content that are rapidly upregulated in response to an inflammatory stimulus. Herein, we review the current knowledge regarding the expression and potential role of MTs in IBD. MTs exert a central position in zinc homeostasis, modulate the activation of the transcription factor nuclear factor (NF)-κB, and serve as antioxidants. In addition, MTs could be involved in IBD through their antiapoptotic effects or through specific immunomodulating extracellular effects. Reports on MT expression in IBD are contradictory but clearly demonstrate a deviant MT expression supporting the idea that these aberrations in IBD require further clarification.

Absence of placental growth factor blocks dextran sodium sulfate-induced colonic mucosal angiogenesis, increases mucosal hypoxia and aggravates acute colonic injury

Angiogenesis has recently been described as a component of inflammatory bowel disease. Placental growth factor (PlGF), a vascular endothelial growth factor (VEGF) homologue, establishes its angiogenic capacity under pathophysiological conditions. We investigated the function of PlGF in experimental models of acute colitis. Acute colonic damage was induced in PlGF knock-out (−/−) mice and PlGF wild-type (+/+) mice by dextran sodium sulfate (DSS) and trinitrobenzenesulfonic acid (TNBS). The concentrations of PlGF and VEGF were measured in distal colonic lysates using an enzyme-linked immunosorbent assay. Colonic injury was evaluated by assessing colon length, colonocyte apoptosis (by terminal dUTP nick-end labeling), colonic cytokine production and histological score. Infiltration of polymorphonuclear cells was determined by assaying myeloperoxidase (MPO) activity. In a separate experiment, recombinant PlGF was administered to PlGF−/− mice by adenoviral transfer before DSS administration. Mucosal vascularization was quantified by computerized morphometric analysis of CD31-stained distal colonic sections. Colonic mucosal hypoxia was visualized by pimonidazole staining. Both VEGF and PlGF were upregulated during acute colitis. In addition, compared with PlGF+/+ controls, PlGF−/− mice showed a significant increase in weight loss and colonic shortening during both DSS and TNBS colitis. This correlated with enhanced colonocyte apoptosis, elevated colonic cytokine levels and increased histological damage score, but not with enhanced inflammatory cell infiltration (MPO activity). The increased morbidity of PlGF−/− mice during DSS colitis was preventable by adenovirus (Ad)-mediated overexpression of PlGF. After the administration of DSS, strongly reduced mucosal angiogenesis was observed in PlGF−/− mice compared with PlGF+/+ mice. This was associated with an early increase in intestinal epithelial pimonidazole accumulation in PlGF−/− mice, suggesting a function of enhanced epithelial hypoxia in the observed differences between the two groups. In summary, our data show that the absence of PlGF strongly inhibits mucosal intestinal angiogenesis in acute colitis, which is associated with an early increase in intestinal epithelial hypoxia and aggravation of the course of the disease.

Staphylococcus aureus Enterotoxin B Regulates Prostaglandin E2 Synthesis, Growth, and Migration in Nasal Tissue Fibroblasts

BACKGROUND Superantigens and eicosanoids are important amplifiers and regulators of inflammation in airway diseases. We therefore studied the possible influence of Staphylococcus aureus enterotoxin B (SEB) on the cyclooxygenase (COX) pathway and basic functions of airway structural cells. METHODS Fibroblasts were isolated from nasal inferior turbinate tissue and cultured in the presence of different concentrations of SEB. Preincubation with interferon (IFN)-gamma was performed to induce expression of major histocompatibility complex (MHC) class II receptors. Prostaglandin E2 (PGE(2)) production was assayed by enzyme-linked immunosorbent assay, and levels of COX-2 and prostanoid E receptors 1-4 (EP(1-4)) were assayed by real-time polymerase chain reaction. Migration and growth tests were performed, and SEB was localized within the cells by confocal microscopy. RESULTS Stimulation with IFN-gamma and SEB significantly down-regulated PGE(2), COX-2, and EP(2) expression but not EP(1), EP(3), or EP(4) expression. The enterotoxin blocked cell growth but increased the fibroblast migration rate. SEB was localized within the cell in the presence and absence of MHC-II, suggesting that mechanisms other than conventional binding may allow the enterotoxin to enter the cell. CONCLUSIONS These findings may have major implications for our understanding of the role played by bacterial superantigens in regulating the inflammatory and remodeling mechanisms of upper airway diseases and hence may help elucidate the pathophysiology of these diseases.

Paracellular Entry of Interleukin-10 Producing Lactococcus lactis in Inflamed Intestinal Mucosa in Mice

Background: Genetically modified Lactococcus lactis secreting interleukin‐10 (IL‐10) has been demonstrated to provide localized delivery of a therapeutic agent through active in situ synthesis in murine colitis. At present, many aspects of the exact mechanism by which the beneficial effect of the IL‐10‐producing L. lactis on the mucosa is mediated remain to be clarified. Methods: Our aim was to determine the interaction of L. lactis with the intestinal mucosa. Therefore, we administered IL‐10‐producing L. lactis to healthy mice and in 2 mouse models of chronic colitis. Paraffin sections of ileum and colon samples were examined with confocal and transmission electron microscopy. Ileum and colon homogenates were prepared after flushing and after removal of mucus layer and epithelium. These homogenates and homogenates of mesenteric lymph nodes and spleen were plated on agar and immunoblotting for L. lactis and IL‐10 was performed. Results: Both confocal and electron microscopy showed the presence of lactococci in inflamed intestinal mucosa of mice with colitis. We recovered viable bacteria that could still produce IL‐10 from homogenates of inflamed ileum and colon of which mucous and epithelial layers were removed. We did not find lactococci in mesenteric lymph nodes or in the spleen of mice with colitis. Conclusions: This study demonstrates uptake of IL‐10‐secreting L. lactis by the paracellular route in inflamed mucosal tissue. We suggest that IL‐10 production by L. lactis residing inside the mucosa in the vicinity of responsive cells can improve the local action of interleukin‐10 in inflamed tissue and the efficiency of the treatment.

Unconditioned adult-derived neurosphere cells mainly differentiate towards astrocytes upon transplantation in sclerotic rat hippocampus

PURPOSE Cell transplantation is being investigated as an alternative treatment for medically refractory temporal lobe epilepsy (TLE). In this study the fate of adult-derived neurosphere cells was evaluated after transplantation in the lesioned hippocampus of the intrahippocampal kainic acid (KA) model for TLE. METHODS Neurosphere-forming cells were derived from the subventricular zone (SVZ) of transgenic green fluorescent protein (GFP) reporter mice and expanded in culture. After 10 passages in vitro neurosphere-derived cells were transplanted in the hippocampus three days (KA3d group) and three weeks (KA3w group) after intrahippocampal KA injection. Survival and differentiation of neurosphere cells were evaluated three and six weeks after transplantation. RESULTS A fraction (about 1%) of GFP-expressing neurosphere cells survived for at least six weeks after transplantation with a higher and more robust survival rate in the KA3d compared to the KA3w group. Although a small fraction of the cells expressed the neuronal marker NeuN, neurosphere cells mainly differentiated towards astrocytes. DISCUSSION Our results indicate that adult-derived neurosphere cells are able to survive upon transplantation in the sclerotic hippocampus. The transplanted cells do not or hardly contribute to neuronal replacement and mainly adopt an astrogliotic fate.