Andrew C. Keates

Development of chronic colitis is dependent on the cytokine MIF

The cytokine macrophage-migration inhibitory factor (MIF) is secreted by a number of cell types upon induction by lipopolysaccharide (LPS). Because colitis is dependent on interplay between the mucosal immune system and intestinal bacteria, we investigated the role of MIF in experimental colitis. MIF-deficient mice failed to develop disease, but reconstitution of MIF-deficient mice with wild-type innate immune cells restored colitis. In addition, established colitis could be treated with anti-MIF immunoglobulins. Thus, murine colitis is dependent on continuous MIF production by the innate immune system. Because we found increased plasma MIF concentrations in patients with Crohns disease, these data suggested that MIF is a new target for intervention in Crohns disease.

cag+ Helicobacter pylori Induce Transactivation of the Epidermal Growth Factor Receptor in AGS Gastric Epithelial Cells

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find thatH. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag+ strains ofH. pylori are able to induce greater EGF receptor activation than cag− strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGF-like growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag+ H. pylori infection.

p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis

Clostridium difficile toxin A causes acute neutrophil infiltration and intestinal mucosal injury. In cultured cells, toxin A inactivates Rho proteins by monoglucosylation. In monocytes, toxin A induces IL-8 production and necrosis by unknown mechanisms. We investigated the role of mitogen-activated protein (MAP) kinases in these events. In THP-1 monocytic cells, toxin A activated the 3 main MAP kinase cascades within 1 to 2 minutes. Activation of p38 was sustained, whereas stimulation of extracellular signal-regulated kinases and c-Jun NH(2)-terminal kinase was transient. Rho glucosylation became evident after 15 minutes. IL-8 gene expression was reduced by 70% by the MEK inhibitor PD98059 and abrogated by the p38 inhibitor SB203580 or by overexpression of dominant-negative mutants of the p38-activating kinases MKK3 and MKK6. SB203580 also blocked monocyte necrosis and IL-1beta release caused by toxin A but not by other toxins. Finally, in mouse ileum, SB203580 prevented toxin A-induced neutrophil recruitment by 92% and villous destruction by 90%. Thus, in monocytes exposed to toxin A, MAP kinase activation appears to precede Rho glucosylation and is required for IL-8 transcription and cell necrosis. p38 MAP kinase also mediates intestinal inflammation and mucosal damage induced by toxin A.

Colonic epithelial cells are a major site of macrophage inflammatory protein 3α (MIP-3α) production in normal colon and inflammatory bowel disease

Background and aim: Macrophage inflammatory protein 3α (MIP-3α) is a recently described lymphocyte directed C-C chemokine expressed predominately at extralymphoid sites, including the intestine. The aim of this study was to determine whether colonic epithelial cells produce MIP-3α and whether its expression is upregulated in inflammatory bowel disease. Methods and results: We found that interleukin 1β and tumour necrosis factor α dose dependently stimulated MIP-3α production in Caco-2 and HT-29 intestinal epithelial cells. In cytokine treated Caco-2 and HT-29 cells, a significant increase in MIP-3α protein production was observed after three hours and continued for at least 24 hours. Analysis of colonic tissues by quantitative real time polymerase chain reaction and ELISA revealed significantly elevated MIP-3α mRNA levels (7.9-fold; p<0.05) and protein levels (8.9-fold; p<0.05) in Crohn’s disease compared with controls or ulcerative colitis. MIP-3α immunoreactivity in normal colon and inflammatory bowel disease was principally associated with crypt and surface epithelial cells. Moreover, MIP-3α protein levels were elevated in primary epithelial cells isolated from patients with inflammatory bowel disease. Conclusions: These findings indicate that increased enterocyte MIP-3α production may play an important role in lymphocyte activation and recruitment to the colonic epithelium in Crohn’s disease and ulcerative colitis.

IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes

Neutrophil infiltration is central to the pathogenesis of Clostridium difficile toxin A-induced enterocolitis. This study examines whether monocyte activation by C. difficile toxins is instrumental in initiating neutrophil activation and recruitment. Human monocytes were exposed to low concentrations of highly purified C. difficile toxins, and the conditioned media were harvested for cytokine and functional assays. Monocytes exposed to C. difficile toxin A (10(-10) M) or toxin B (10(-12) M) released 100 and 20 times basal levels, respectively, of the neutrophil chemoattractant interleukin-8 (IL-8). Reverse transcriptase-polymerase chain reaction demonstrated a marked increase in IL-8 mRNA expression by monocytes 3 h after toxin exposure. Conditioned media from toxin A- and toxin B-treated monocytes stimulated neutrophil migration (324 and 245% of control, respectively). This effect was completely blocked by IL-8 antiserum. These media also upregulated neutrophil CD11b/CD18 and endothelial cell intercellular adhesion molecule-1 expression. C. difficile toxins, at low concentrations, potently activate monocytes to release factors, including IL-8, that facilitate neutrophil extravasation and tissue infiltration. Our findings indicate a major role for toxin-mediated monocyte and macrophage activation in C. difficile colitis.

Ionizing Radiation Induces Stemness in Cancer Cells

The cancer stem cell (CSC) model posits the presence of a small number of CSCs in the heterogeneous cancer cell population that are ultimately responsible for tumor initiation, as well as cancer recurrence and metastasis. CSCs have been isolated from a variety of human cancers and are able to generate a hierarchical and heterogeneous cancer cell population. CSCs are also resistant to conventional chemo- and radio-therapies. Here we report that ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. Exposure of non-stem cancer cells to ionizing radiation enhanced spherogenesis, and this was accompanied by upregulation of the pluripotency genes Sox2 and Oct3/4. Knockdown of Sox2 or Oct3/4 inhibited radiation–induced spherogenesis and increased cellular sensitivity to radiation. These data demonstrate that ionizing radiation can activate stemness pathways in heterogeneous cancer cells, resulting in the enrichment of a CSC subpopulation with higher resistance to radiotherapy.

P‐Glycoprotein‐170 inhibition significantly reduces cortisol and ciclosporin efflux from human intestinal epithelial cells and T lymphocytes

To assess the role of P‐glycoprotein‐170 (P‐gp) in transporting cortisol and ciclosporin from human intestinal epithelium and T lymphocytes.

CGRP upregulation in dorsal root ganglia and ileal mucosa during Clostridium difficile toxin A-induced enteritis

We have previously reported that pretreatment of rats with capsaicin (an agent that ablates sensory neurons) or CP-96345 (a substance P receptor antagonist) dramatically inhibits fluid secretion and intestinal inflammation in ileal loops exposed to Clostridium difficile toxin A. The aim of this study was to determine whether calcitonin gene-related peptide (CGRP), a neuropeptide also found in sensory afferent neurons, participates in the enterotoxic effects of toxin A. Administration of toxin A was also found to increase CGRP content in dorsal root ganglia and ileal mucosa 60 min after toxin exposure. Furthermore, immunohistochemical studies demonstrated increased neuronal staining for CGRP 2 h after toxin A treatment. Pretreatment of rats with CGRP-(8-37), a specific CGRP antagonist, before instillation of toxin A into ileal loops significantly inhibited toxin-mediated fluid secretion (by 48%), mannitol permeability (by 83%), and histological damage. We conclude that CGRP, like substance P, contributes to the secretory and inflammatory effects of toxin A via increased production of this peptide from intestinal nerves, including primary sensory afferent neurons.We have previously reported that pretreatment of rats with capsaicin (an agent that ablates sensory neurons) or CP-96345 (a substance P receptor antagonist) dramatically inhibits fluid secretion and intestinal inflammation in ileal loops exposed to Clostridium difficile toxin A. The aim of this study was to determine whether calcitonin gene-related peptide (CGRP), a neuropeptide also found in sensory afferent neurons, participates in the enterotoxic effects of toxin A. Administration of toxin A was also found to increase CGRP content in dorsal root ganglia and ileal mucosa 60 min after toxin exposure. Furthermore, immunohistochemical studies demonstrated increased neuronal staining for CGRP 2 h after toxin A treatment. Pretreatment of rats with CGRP-(8-37), a specific CGRP antagonist, before instillation of toxin A into ileal loops significantly inhibited toxin-mediated fluid secretion (by 48%), mannitol permeability (by 83%), and histological damage. We conclude that CGRP, like substance P, contributes to the secretory and inflammatory effects of toxin A via increased production of this peptide from intestinal nerves, including primary sensory afferent neurons.

Transactivation of the epidermal growth factor receptor by cag+ Helicobacter pylori induces upregulation of the early growth response gene Egr-1 in gastric epithelial cells

Background and aims:Helicobacter pylori, in particular cytotoxin associated gene (cag)+ strains, have been shown to enhance gastric epithelial cell proliferation in vivo, an effect that likely contributes to gastric carcinogenesis. Early growth response gene 1 (Egr-1) is a crucial regulator of cell growth, differentiation, and survival, which is known to play a role in carcinogenesis and cancer progression. The aims of this study were to: (1) examine whether H pylori could upregulate Egr-1 in gastric epithelial cell lines; (2) determine whether there was a differential response to infection with different strains; (3) examine the role of the cag pathogenicity island in this process; and (4) elucidate the molecular mechanisms leading to Egr-1 upregulation. Methods and results: We found that infection of AGS cells with cag+H pylori resulted in a rapid (1–2 hours) but transient increase in Egr-1 mRNA and protein levels whereas coculture with cag− isolates did not elicit this response. Furthermore, two independent cagE− isogenic mutants of H pylori also demonstrated impaired ability to upregulate Egr-1. Upregulation of Egr-1 protein was inhibited by the extracellular regulated kinase (ERK)1/2 inhibitor PD98059 and overexpression of dominant negative MEK1 downregulated Egr-1 luciferase reporter gene activity. Treatment of AGS cells with the epidermal growth factor receptor (EGFR) kinase inhibitors PD153035 and AG1478 resulted in a reduction in H pylori mediated Egr-1 upregulation, demonstrating that EGFR transactivation plays a role in this early cellular process. Conclusions: Our findings show that cag+H pylori cause rapid induction of Egr-1 in gastric epithelial cells which may contribute to H pylori mediated pathogenesis.

Human Papillomavirus Type 8 Interferes with a Novel C/EBPβ-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration

Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBPβ converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBPβ as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBPβ in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBPβ-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.