Christina Gustafson-Svärd

Cyclooxygenase-1 and cyclooxygenase-2 gene expression in human colorectal adenocarcinomas and in azoxymethane induced colonic tumours in rats.

Increased prostaglandin E2 synthesis is considered important in both human and experimental colon carcinogenesis. It is not known, however, which cyclooxygenase isoenzyme is involved. The aim of this study was to compare the content of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in colorectal cancers with the content in normal colonic specimens. Fifteen human colorectal adenocarcinomas, 35 azoxymethane induced colonic tumours from rats, and specimens of normal colon were analysed by reverse transcription and polymerase chain reaction (RT-PCR). It was found that cyclooxygenase-1 and cyclooxygenase-2 mRNA were increased in azoxymethane induced colonic tumours, compared with specimens taken adjacent to the tumours or from the macroscopically normal intestine distant from the tumours. Cyclooxygenase-1 and cyclooxygenase-2 mRNA were increased in specimens from the macroscopically normal intestine of azoxymethane treated animals, compared with colonic specimens from saline treated rats. Cyclooxygenase-2 mRNA, but not cyclooxygenase-1 mRNA, was increased in human colorectal cancers, compared with the adjacent mucosa or macroscopically normal mucosa distant from the tumours. The results suggest that cyclooxygenase-2 is involved in the increased prostaglandin E2 synthesis in colonic cancers, and that activation of this isoenzyme is an early event in colon carcinogenesis. However, cyclooxygenase-1 may also be involved, at least in experimental colon carcinogenesis.

Tumor necrosis factor-alpha in ileal mast cells in patients with Crohn's disease.

Background: Reports that both intestinal and extraintestinal Crohn’s disease (CD) had healed successfully after treatment with anti-tumor necrosis factor-alpha (TNF-α) antibody have strengthened the hypothesis that it has a role in the treatment of CD. The macrophage is one source of TNF-α. Intestinal mast cells are also thought to have a role in CD, but it is not known if human ileal mast cells express TNF-α. Aim: To find out whether TNF-α is expressed by mast cells in the ileal wall in CD patients and controls. Methods: TNF-α was sought immunohistochemically in full thickness specimens of ileal wall from patients with CD (histologically normal, n = 9; inflamed, n = 6) and controls (patients with colonic cancer, n = 8). Mast cells were identified by metachromasia and anti-mast cell tryptase immunoreactivity. Results: In all layers of the ileal wall, and in every specimen investigated, mast cells were the main cell type that expressed TNF-α immunoreactivity out of the TNF-α-labelled cells. The number of TNF-α- labelled mast cells was greater in the muscularis propria in patients compared with controls, both in uninflamed (1.7-fold, p < 0.05) and in inflamed bowel (4.6-fold, p < 0.002); greater in the submucosa in inflamed compared with uninflamed CD (1.6-fold, p < 0.01), and less in the lamina propria in inflamed compared with uninflamed CD (0.4-fold, p < 0.05). Conclusion: Mast cells are an important source of TNF-α in all layers of the ileal wall, and the increased density of TNF-α-positive mast cells in the submucosa and muscularis propria may contribute to the tissue changes and symptoms in CD.

Phospholipase A2 gene expression and activity in histologically normal ileal mucosa and in Crohn's ileitis.

Increased activity of phospholipase A2 (PLA2) in the ileal mucosa may contribute to the inflammation in Crohns disease. The results of this study showed that (a) three months after ileocolonic resection for Crohns disease the neoterminal ileal mucosa showed endoscopically new inflammation and had higher PLA2 activity than at the time of the operation (n = 8); no such findings were seen in controls (n = 7), (b) histologically normal ileal mucosa (n = 3) contained mRNA for three isoforms of PLA2 (PLA2-I, PLA2-II, and cPLA2), but the amounts of PLA2-II mRNA clearly exceeded the amounts of mRNA for PLA2-I and cPLA2, (c) ileal mucosa from Crohns patients (n = 2) contained higher values of PLA2-II mRNA than ileal mucosa from two controls, (d) ileal mucosa from Crohns patients (n = 4) showed increased PLA2-II mRNA three months after ileocolonic resection. In conclusion, these results show that the predominating PLA2 mRNA in the human ileal mucosa is type II PLA2, and the increased synthesis of PLA2-II might be responsible for the increased PLA2 activity found in the ileal mucosa accompanying recurrent ileal inflammation in Crohns disease.

Tumor Necrosis Factor-α Potentiates Phospholipase A2-Stimulated Release and Metabolism of Arachidonic Acid in Cultured Intestinal Epithelial Cells (INT 407)

Tumor necrosis factor-alpha (TNF-alpha), a known pro-inflammatory cytokine, has been suggested to play a role in the pathogenesis of inflammatory bowel disease (IBD) by mediating damage to the intestinal epithelial cells. The present study demonstrates that TNF-alpha potentiates release and metabolism of 14C-labeled arachidonic acid (14C-AA) in cultured intestinal epithelial cells (INT 407). Although TNF-alpha on its own was but a weak stimulator of cellular 14C-AA turnover, it significantly potentiated the release of 14C-AA and 14C-labeled prostaglandin E2(14C-PGE2) after stimulation with three known phospholipase A2 activators: phospholipase. C from Clostridium perfringens, the calcium ionophore A23187, and the phorbol ester 4-beta-phorbol-12-myristate-13-acetate (PMA). The phospholipase A2 inhibitor quinacrine significantly reduced both AA and PGE2 release after combined stimulation with phospholipase C and TNF-alpha. In contrast to its effect on the AA turnover, TNF-alpha did not affect the phospholipase C-stimulated production of platelet-activating factor (PAF-acether). Taken together, these findings indicate that a) TNF-alpha potentiates phospholipase A2-stimulated AA release from cultured intestinal epithelial cells; b) TNF-alpha may stimulate phospholipase A2-dependent AA release without affecting the formation of PAF-acether and c) pretreatment with TNF-alpha potentiates the formation of PGE2 after stimulation with phospholipase A2 activators. In summary, the present investigation points to the possibility that TNF-alpha may stimulate intestinal epithelial cells to produce biologically active AA metabolites and that this stimulation may be modulated by components of the intestinal luminal content, like bacterial toxins.

Presence of group IIa secretory phospholipase A2 in mast cells and macrophages in normal human ileal submucosa and in Crohn's disease

Abstract Secretory group IIa phospholipase A2 (PLA2-II) is an important regulator of proinflammatory lipid mediator production and may play a role in ileal inflammation in Crohns disease. The enzyme has previously only been detected in epithelial Paneth cells. However, one characteristic feature of Crohns disease is the transmural inflammation. Full thickness ileal sections from nine patients with Crohns disease, and histologically normal sections from patients with colonic cancer (n=7) and chronic severe constipation (n=1) as controls, were used in this study. PLA2-II-positive cells were detected by immunofluorescence and in situ hybridization. Metachromatic staining and esterase staining were used to identify mast cells and macrophages, respectively. It was shown that mast cells and macrophages in the ileal submucosa in both patients and controls showed positive PLA2-II staining. The number of PLA2-II-labeled cells that did not react with metachromasia, e.g. macrophages, was significantly greater in inflamed Crohns disease compared to controls. This is, to our knowledge, the first study that has described the presence in healthy, while presence and upregulation of PLA2-II-positive cells in inflamed human ileal submucosa. Our findings suggest a proinflammatory potential for secretory PLA2-II in submucosa, while proinflammatory stimulation of mast cells and macrophages in vitro has shown that the enzyme is responsible for delayed prostaglandin formation.

Cyclo-oxygenase and Colon Cancer: Clues to the Aspirin Effect?

Epidemiological and experimental studies indicate an inverse relationship between the risk of colon cancer development and intake of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). All NSAIDs are known inhibitors of cyclo-oxygenase, the enzyme responsible for converting arachidonic acid to prostaglandins. Prostaglandins have been implicated in the pathogenesis of colon cancer and it has been suggested that the preventive effect of NSAIDs is due to inhibition of cyclo-oxygenase activity. Cyclo-oxygenase exists in two different isoforms, cyclo-oxygenase-1 and cyclo-oxygenase-2, and data obtained during the last few years have suggested that cyclo-oxygenase-2 might be involved in both human and experimental colon carcinogenesis. The purpose of this review is to provide an update on recent studies regarding cyclo-oxygenase, in particular cyclo-oxygenase-2, in relation to colon cancer in humans and in experimental models.

Effects of endotoxin and dexamethasone on group I and II phospholipase A2 in rat ileum and stomach.

Phospholipase A2 (EC 3.1.1.4) is a key enzyme in inflammation and is thought to play an important part in inflammatory diseases of the gastrointestinal tract. To investigate the nature and regulation of phospholipase A2 activity in the gastrointestinal mucosa, the distribution of messenger ribonucleic acid (mRNA) for group II phospholipase A2 in various parts of the rat gastrointestinal tract was studied, as well as the influence of endotoxin or dexamethasone, or both, on the group I and II phospholipase A2 mRNA expression and activity in the rat glandular stomach and distal ileum. The results show that (a) group II phospholipase A2 is present along the whole gastrointestinal tract, but in particularly large amounts in the distal ileum, (b) endotoxin increases group II, but not group I, phospholipase A2 mRNA expression in the glandular stomach and distal ileum, and (c) dexamethasone reduces the endotoxin induced increases in group II phospholipase mRNA expression and activity in the gastrointestinal mucosa. These findings suggest that phospholipase A2 of type II is a mediator of endotoxin effects in the gastrointestinal mucosa and that its expression at the mRNA level can be inhibited by corticosteroids.

Cytosolic phospholipase A2 and cyclooxygenase-2 mediate release and metabolism of arachidonic acid in tumor necrosis factor-α-primed cultured intestinal epithelial cells (INT 407)

BACKGROUND We have recently reported that tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine that has been suggested to play a role in the pathogenesis of inflammatory bowel disease, potentiates phospholipase A2 (PLA2)-stimulated arachidonic acid (AA) release and prostaglandin E2 (PGE2) formation in cultured intestinal epithelial cells (INT 407). The aim of the present study was to investigate which particular isoforms of PLA2 and cyclooxygenase (COX) are involved in these processes. METHODS Cells were labeled with 14C-AA or 14C-oleic acid, and the amounts of released fatty acid and PGE2 were analyzed by thin-layer chromatography. mRNA was analyzed by reverse transcription and polymerase chain reaction. RESULTS The cells contained mainly mRNA for cytosolic PLA2 (cPLA2) and only trace amounts of mRNA for group I and II PLA2. TNF-alpha potentiated the release of 14C-AA but not of 14C-oleic acid. The TNF-alpha-potentiated PGE2 release was reduced after inhibition of cellular COX activity or mRNA synthesis. TNF-alpha increased the amounts of mRNA for COX-2 but not for COX-1. CONCLUSIONS The results point to the possibility that TNF-alpha may modulate the intestinal mucosal content of biologically active AA metabolites by priming cPLA2- and COX-2-mediated processes in the epithelial cells.

Group I phospholipase A2 mRNA expression in rat glandular stomach and pancreas : Ontogenic development and effects of cortisone acetate.

The postnatal development of group I phospholipase A2 (group I PLA2) in the glandular stomach and pancreas of neonatal rats was investigated. The amounts of group I PLA2 mRNA (and also the PLA2 enzymatic activity) in the glandular stomach mucosa increased with age in 3-60-day-old animals. This postnatal development of rat stomach group I PLA2 mRNA agreed with that of group I PLA2 mRNA of the rat pancreas, and thus seems to follow the general development of the gastrointestinal tract during the neonatal period. The latter was further supported by the finding that maturation of group I PLA2 in both the stomach and pancreas was induced precociously in rats treated with cortisone acetate. It is suggested that the stomach group I PLA2 is involved in mucosal eicosanoid production.

Phospholipase C from Clostridium perfringens stimulates acetyltransferase-dependent formation of platelet-activating factor in cultured intestinal epithelial cells (INT 407)

The mechanisms by which phospholipase C from Clostridium perfringens stimulates the formation of platelet-activating factor (PAF-acether) in cultured intestinal epithelial cells (INT 407) were investigated. Although stimulation with phospholipase C caused a significant formation of PAF-acether, there was no significant increase in the cellular levels of lysoPAF-acether after stimulation. Moreover, when cells prelabeled with 3H-1-O-alkyl-2-acyl-sn-glycerophosphocholine were stimulated with phospholipase C, the 3H-lysoPAF-acether content was not increased in stimulated cells as compared with unstimulated cells. When cells were preincubated with the calmodulin inhibitor trifluoperazine (TFPA), the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), or the combined phospholipase A2-inhibitor and lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) before stimulation with phospholipase C, the PAF-acether formation was significantly decreased. The phospholipase A2 inhibitor 4-bromophenacyl bromide (BPB), on the other hand, had no significant effect on the PAF-acether formation. Preincubation with NDGA also decreased the levels of lysoPAF-acether, whereas BPB, H7, or TFPA had no such effect. These findings indicate that stimulation of acetyltransferase activity with increased acetylation of lysoPAF-acether may be one way by which phospholipase C from C. perfringens stimulates formation of PAF-acether in INT 407 cells.