Fadia R. Homaidan

Human T-cell lymphotropic virus type I-infected cells extravasate through the endothelial barrier by a local angiogenesis-like mechanism

Extravasation of tumor cells through the endothelial barrier is a critical step in cancer metastasis. Human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia/lymphoma (ATL) is an aggressive disease characterized by visceral invasion. We show that ATL and HTLV-I-associated myelopathy patients exhibit high plasma levels of functional vascular endothelial growth factor and basic fibroblast growth factor. The viral oncoprotein Tax transactivates the promoter of the gap-junction protein connexin-43 and enhances gap-junction-mediated heterocellular communication with endothelial cells. The interaction of HTLV-I-transformed cells with endothelial cells induces the gelatinase activity of matrix metalloproteinase (MMP)-2 and MMP-9 in endothelial cells and down-regulates the tissue inhibitor of MMP. This leads to subendothelial basement membrane degradation followed by endothelial cell retraction, allowing neoplastic lymphocyte extravasation. We propose a model that offers a mechanistic explanation for extravasation of HTLV-I-infected cells: after specific adhesion to endothelia of target organs, tumor cells induce a local and transient angiogenesis-like mechanism through paracrine stimulation and direct cell-cell communication with endothelial cells. This culminates in a breach of the endothelial barrier function, allowing cancer cell invasion. This local and transient angiogenesis-like sequence that may facilitate visceral invasion in ATL represents a potential target for ATL therapy.

Protein regulators of eicosanoid synthesis: role in inflammation.

A variety of factors contribute to the complex course of inflammation. Microbiological, immunological and toxic agents can initiate the inflammatory response by activating a variety of humoral and cellular mediators. In the early phase of inflammation, excessive amounts of cytokines and inflammatory mediators are released. These factors activate, in addition to other signaling pathways, the lipid synthesis pathways, which play a crucial role in the pathogenesis of organ dysfunction. Arachidonic acid (AA), the precursor of pro-inflammatory eicosanoids, is released from membrane phospholipids by the action of phospholipase A(2) (PLA(2)), and is metabolized to prostaglandins (PGs) and leukotrienes (LTs) by the action of cyclooxygenase (COX) and lipoxygenase (LO) enzymes, respectively. Disordered activation of PLA(2), LO and COX enzymes have been implicated in many inflammatory diseases. PLA(2) is activated by phospholipase-A(2)-activating protein (PLAP) and LO by 5-lipoxygenase-activating protein (FLAP). The inducible form of COX-2 enzyme, which is usually not present under basal conditions, is induced in inflammation. In this article the function of these enzymes in eicosanoid synthesis, their regulation, and their implication in inflammatory disorders will be reviewed. The properties, function and regulation of the protein activators PLAP and FLAP will also be discussed.

Adhesion and Cytosolic Dye Transfer between Macrophages and Intestinal Epithelial Cells

Activated macrophages (M phi) found in the intestinal lesions of patients with inflammatory bowel disease (IBD) secrete many inflammatory mediators which can regulate intestinal epithelial cell (IEC) function. However, little is known about direct M phi-IEC interactions. Two potential mechanisms by which cells may interact are through specific receptor-ligand binding of adhesion molecules, such as integrins or cadherins, and by exchange of cytoplasmic substances through transmembraneous channels called gap junctions. We investigated whether M phi could adhere to epithelial cells in culture and form transmembrane communication channels as defined by dye transfer. Primary cultures of murine M phi and a M phi cell line, P388D1, adhered to Mode-K and IEC6, but not CMT-93 IEC. Antibody blocking studies determined that P388D1-Mode-K binding was partially dependent on beta 2 integrin (CD18) function, Mode-K constitutively expressed CD106 (VCAM-1) and cell associated fibronectin, while P388D1 expressed low levels of CD49d/CD29 (VLA4) but blocking antibodies to these surface molecules did not inhibit P388D1-Mode-K adherence. Transfer of calcein dye from M phi to IEC was quantitated by flow cytometry and was dependent on M phi-IEC adhesion. Dye transfer was concentration dependent in that the fluorescence intensity of Mode-K was proportional to the number of adherent P388D1 cells as well as the dye load of the M phi. These results indicate that M phi interact with IEC by adhesion and possibly through gap junctions and may thus regulate IEC function by direct cell-cell communication.

Gap junctional communication between murine macrophages and intestinal epithelial cell lines.

In intestinal inflammation, inflammatory cells infiltrate the submucosa and are found juxtaposed to intestinal epithelial cell (IEC) basolateral membranes and may directly regulate IEC function. In this study we determined whether macrophage (M phi), P388D1 and J774A.1, are coupled by gap junctions to IEC lines, Mode-K and IEC6. Using flow cytometric analysis, we show bi-directional transfer of the fluorescent dye, calcein (700 Da) between IEC and M phi resulting in a 3.5-20-fold increase in recipient cell fluorescence. Homocellular and heterocellular dye transfer between M phi and/or IEC was detected in cocultures of P388D1, J774A.1, Mode-K, IEC6 and CMT93. However, transfer between P388D1 and Mode-K was asymmetrical in that transfer from P388D1 to Mode-K was always more efficient than transfer from Mode-K to P388D1. Dye transfer was strictly dependent on IEC-M phi adhesion which in turn was dependent on the polarity of IEC adhesion molecule expression. Both calcein dye transfer and adhesion were inhibited by the addition of heptanol to cocultures. Furthermore we demonstrate both IEC homocellular, and M phi-IEC heterocellular propagation of calcium waves in response to mechanical stimulation, typical of gap junctional communication. Finally, areas of close membrane apposition were seen in electron micrographs of IEC-M phi cocultures, suggestive of gap junction formation. These data indicate that IEC and M phi are coupled by gap junctions suggesting that gap junctional communication may provide a means by which inflammatory cells might regulate IEC function.

Regulation of nuclear factor-kappaB in intestinal epithelial cells in a cell model of inflammation.

BACKGROUND: Interleukin-1 (IL-1), an inflammatory cytokine whose levels are elevated in inflamed mucosa, causes part of its effect on intestinal epithelial cells (IEC) through inducing ceramide production. AIM: To study the role of nuclear factor-kappaB (NF-kappaB), a pro-inflammatory and anti-apoptotic factor, in IL-1-treated IEC. METHODS: NF-kappaB activity and levels of apoptotic proteins were assessed by electrophoretic mobility shift assay and RNA-protection assay, respectively. RESULTS: IL-1 and ceramide, which have been shown to partially mediate IL-1 effects on IEC, activated NF-kappaB levels significantly. This activation was due to a decrease in IkappaB-alpha and IkappaB-beta protein levels. Moreover, the ratio of mRNA levels of anti-apoptotic to pro-apoptotic proteins was significantly increased in IL-1-treated IEC. CONCLUSION: NF-kappaB may play a key role in the regulation of the expression of pro-inflammatory and/or apoptotic genes in inflammatory bowel disease, making this protein an attractive target for therapeutic intervention.

Anti-Inflammatory properties of Salograviolide A purified from Lebanese plant Centaurea ainetensis

BackgroundAnti-inflammatory activities of medicinal plants have largely been attributed to their content of sesquiterpene lactones (SLs). SLs are predominantly found in the sunflower family Asteraceae and have been isolated from many plants of this family, particularly Centaurea. The anti-inflammatory activities of extract of Centaurea ainetensis, a Lebanese endemic plant, and the isolated active molecule were assessed for their potential ant-inflammatory activities.MethodsPlant extract from Centaurea ainetensis, and the isolated active ingredient Salograviolide A (SA), a sesquiterpene lactones guaianolide, were used for the study. Western blotting and electrophoretic mobility shift assays were used to test the effects of the plant extract and SA on interleukin-1 (IL-1) induced increase in cyclooxygenase-2 (COX-2) levels and in nuclear factor-κB (NF-κB) translocation in an intestinal epithelial cell (IEC) of inflammation. Their effects on inflammation score and cytokine levels were also studied in an iodoacetoamide-induced rat model of inflammation.ResultsPlant extract and SA were shown to reverse the effects observed by IL-1 on COX-2 levels and NF-κB translocation in IEC. SA decreased the level of inflammatory cytokines and the level of inflammation in the animal model.ConclusionThese findings suggest that SA may be useful in the development of natural therapies for inflammatory diseases.

Regulation of ion transport by histamine in mouse cecum

Histamine levels are elevated in inflammatory bowel disease. We investigated the mechanism by which histamine affects electrolyte transport in the mouse cecum. Using the Ussing-chamber voltage clamp technique, histamine was found to cause a transient concentration-dependent increase in short-circuit current, a measure of total ion transport across the epithelial tissue. This increase was not affected by amiloride pretreatment, but was significantly inhibited by bumetanide and completely inhibited when chloride was substituted in the bathing buffer by gluconate. A histamine-induced increase in short-circuit current was also significantly reduced by inhibitors of the cyclooxygenase pathway indicating the involvement of prostaglandin E2 in its action. Prostaglandin E2 levels were increased in histamine treated tissue and this increase was reversed by indomethacin. These data suggest that histamine causes its effect on mouse cecum largely through increasing arachidonic acid metabolism resulting in increased levels of prostaglandins which in turn increase Cl- secretion in the epithelial cells.

IL-1 stimulates ceramide accumulation without inducing apoptosis in intestinal epithelial cells

BACKGROUND: In inflammatory bowel disease (IBD), cytokine levels (such as interleukin-1 (IL-1)) are elevated. We have shown previously that IL-1 activates phospholipid signaling pathways in intestinal epithelial cells (EEC), leading to increased ceramide levels. AIM: To determine whether ceramide induces apoptosis in IEC. METHODS: Apoptosis was evaluated by annexin-V binding or Hoechst nuclear staining. Levels of bcl-2, bcl-x, bax, p53 and p21 were determined by Western blotting, and celi cycle analysis was determined by flow cytometry. RESULTS: IL-1 increased ceramide accumulation in a time-dependent and concentration-dependent manner with a peak response at 4 h, with [IL-1] = 30 ng/ml. Neither IL-1 nor ceramide induced apoptosis in EEC, but they increased bcl-2 levels and decreased bax and p21 levels without affecting bcl-x and p53 levels. They also caused a slight but significant increase in the G2/M phase. These data suggest a role for ceramide in IBD and suggest a possible mechanism for the enhanced tumorigenic activity in IBD patients.

IL-1 activates two phospholipid signaling pathways in intestinal epithelial cells

Abstract.Objective and Design: the aim of the study was to decipher the molecular signals involved in IL-1s action on intestinal epithelial cells (IEC).¶Materials and Methods: Mode-K cells, used as a model of IEC, were treated with IL-1, and PLA2 activity and PGE2, ceramide, and cyclooxygenase-2 (COX-2) levels were measured using enzyme-immuno-assay kit, EIA, thin-layer chromatography and western blotting assays respectively.¶Results: IL-1 caused a concentration- and time-dependent increase in PLA2 activity (3-fold increase), in ceramide levels (peak increase = 10.5 ± 0.9 pmol/nmol phosphate), and in COX-2 and PGE2 levels. PGE2 increase was biphasic with an early peak at 10 min (around 5 ng/mg protein) due to increased PLA2 activity. The later peak (13.1 ± 1.9 ng/mg protein) at 4 h was due to COX-2 induction.¶Conclusion: In conclusion, these findings demonstrate that IL-1 regulates IEC function through two pathways, the PLA2 and the sphingomyelin pathways, both of which are capable of modulating the inflammatory process.

The mechanisms of action of interleukin-1 on rabbit intestinal epithelial cells.

Interleukin-1 (IL-1) is an inflammatory mediator that increases Cl- secretion in intestinal epithelial cells. To identify the signal transduction pathway(s) involved in IL-1s action, cells were treated with IL-1 and the levels of cyclooxygenase (COX) enzymes, prostaglandin E2 (PGE2) and phospholipase A2-activating protein (PLAP), and the activity of phospholipase A2 (PLA2) were measured. IL-1 caused concentration- and time-dependent increases in the levels of PLA2 activity, and/or in the levels of PLAP, COX-2 and PGE2. The IL-induced increase in PGE2 levels was biphasic, with the first peak due to the increase in PLAP levels, and the second peak due to the increase in COX-2 levels. This increase in PGE2 levels may provide a mechanism for acute and chronic inflammation in the intestine.