Blocking fcα receptor I on granulocytes prevents tissue damage induced by IgA autoantibodies

IgA represents the most prominent Ab class at mucosal surfaces and the second most prevalent Ab in human blood after IgG. We recently demonstrated that cross-linking of the granulocyte IgA FcR (FcαRI) by IgA induces a chemotactic-driven positive-feedback migration loop, hereby amplifying recruitment of granulocytes to IgA deposits. Therefore, we postulated that aberrant IgA-Ag complexes, which can be found in tissues in IgA-mediated diseases, are responsible for tissue damage by inducing continuous granulocyte migration and activation. Using an IgA-dependent skin-blistering disease as a model system, we demonstrated colocalization of FcαRI-positive granulocyte infiltrates with IgA in cryosections of lesional skin of patients suffering from this disease. Furthermore, we showed granulocyte migration to IgA deposits injected in human skin explants and in murine skin of FcαRI transgenic mice in vivo. Importantly, ex vivo migration and tissue damage were inhibited by blocking FcαRI, indicating that these events are dependent on the interaction of IgA autoantibodies with FcαRI. Thus, interrupting the granulocyte migration loop by blocking FcαRI reduces tissue damage in diseases with aberrant IgA-immune complexes. As such, our results may lead to development of new therapies for IgA-mediated chronic inflammatory diseases, hereby decreasing severe morbidity and improving quality of life for these patients.     

Induction of apoptosis in mouse brain capillary endothelial cells by cyclosporin A and tacrolimus

Although cyclosporin A and tacrolimus are used clinically as potent immunosuppressants, there have been reports of neurotoxicity and encephalopathy. A possible mechanism is that these drugs damage the blood-brain barrier (BBB), inducing dysfunction and increased permeability, and are then able to enter the brain. We studied the cytotoxicity of cyclosporin A and tacrolimus, focused on apoptosis induction, using an immortalized cell line established from BALB/c mouse cerebral microvessel endothelial cells (MBEC4). We found that these two drugs induced cell shrinkage, chromatin condensation and DNA fragmentation, which are characteristics of apoptosis. Our data suggest that the induction of apoptosis on the brain capillary endothelial cells may be at least partly involved in the occurrence of immunosuppressant-induced encephalopathy.     

Epigenetic regulation of extracellular-superoxide dismutase in human monocytes

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall and plays an important role in normal redox homeostasis. We previously showed the significant reduction or induction of EC-SOD during human monocytic U937 or THP-1 cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively; however, its cell-specific expression and regulation have not been fully elucidated. It has been reported that epigenetic factors, such as DNA methylation and histone modification, are involved in several kinds of gene regulation. In this study, we investigated the involvement of epigenetic factors in EC-SOD expression and determined high levels of DNA methylation within promoter and coding regions of EC-SOD in THP-1 cells compared to those in U937 cells. Moreover, treatment with a DNA methyltransferase inhibitor, 5-azacytidine, significantly induced the expression of EC-SOD in THP-1 cells, indicating the importance of DNA methylation in the suppression of EC-SOD expression; however, the DNA methylation status did not change during THP-1 cell differentiation induced by TPA. On the other hand, we detected histone H3 and H4 acetylation during differentiation. Further, pretreatment with histone acetyltransferase inhibitors, CPTH2 or garcinol, significantly suppressed the TPA-inducible EC-SOD expression. We also determined the epigenetic suppression of EC-SOD in peripheral blood mononuclear cells. Treatment with granulocyte macrophage colony-stimulating factor (GM-CSF)/granulocyte-CSF induced that expression. Overall, these findings provide novel evidence that cell-specific and TPA-inducible EC-SOD expression are regulated by DNA methylation and histone H3 and H4 acetylation in human monocytic cells.     

Endotoxin-mediated pulmonary endothelial cell injury

Infusion of endotoxin into sheep results in physiological and structural damage to the pulmonary endothelium. It is uncertain whether complement activation and granulocyte sequestration in the pulmonary microcirculation and the ensuing granulocyte migration into the interstitium seen with endotoxemia contribute to the endothelial damage. We have shown that infusion of complement-activated plasma into sheep, although causing the same degree of granulocyte sequestration in the lungs, results in only modest and transient endothelial damage. In addition, migration or chemotaxis of granulocytes across the endothelial layer of intimal explants is not accompanied by either structural evidence of endothelial damage or a detectable increase in vascular permeability. Such studies indicate that neither complement/granulocyte activation nor granulocyte migration across a vessel wall is entirely responsible for the severe endothelial damage seen with endotoxin. In vitro studies of bovine pulmonary endothelial monolayers indicate that endotoxin can cause direct damage to the endothelium; the damage is dose-dependent and more severe in the presence of serum. Structural studies show endothelial cell retraction, pyknosis, and sloughing. Prostacyclin production and lactic dehydrogenase release are increased, as are permeability to small solutes and hydraulic conductance across the endothelium. It seems that endotoxin can cause a direct injury to pulmonary endothelium but complement and granulocyte activation may enhance the damage.     

Capillary plexus development in the day five to day six chick chorioallantoic membrane is inhibited by cytochalasin D and suramin

The chick chorioallantoic membrane (CAM) is a valuable model for evaluating angiogenesis and vasculogenesis. Our purpose was to characterize the formation of the CAM vasculature, in particular the capillary plexus, between days five and six after fertilization and to examine the mode of action of cytochalasin D and suramin on vascular development during this interval. The CAM increased 20-fold in size between days five and six, during which time the capillary plexus forms by both migration of mesodermal blood vessels toward the ectoderm and by the formation of new vessels from angioblasts near the ectoderm. Between days five and six, the CAM becomes thinner, and the density of the mesodermal cells decreases. To determine the mode of action of anti-angiogenic drugs on the day five to day six CAM, various concentrations of cytochalasin D or suramin were added directly to day five CAMs, and their effects were evaluated on day six. Both drugs significantly inhibited CAM growth, altered branching patterns of the major vessels, decreased area of the major vessels, and inhibited the formation of the capillary plexus by inhibiting both vasculogenesis and the migration of mesodermal blood vessels to the ectoderm. Cytochalasin D also inhibited compartmentalization of the plexus. Cytochalasin D and suramin were inhibitory at similar doses. This study provides new information on early CAM development, establishes the mode of action and dose dependency of cytochalasin D and suramin on day five to day six CAMs, and demonstrates that the day five to day six CAM provides a useful assay to examine the effect of anti-angiogenic drugs on blood vessel development, including capillary plexus formation.     

Induction of endothelial cell migration by proline analogs and its relevance to angiogenesis

The proline analogs cis-4-hydroxy-L-proline, 3,4-dehydro-L-proline, and 2-azetidinecarboxylic acid induced increases in the migration rate of retinal capillary endothelial cells, aortal endothelial cells, corneal endothelial cells, aortal smooth muscle cells, and retinal pericytes. cis-4-hydroxy-D-proline did not. The optimal concentration for migration induction by any of the active agents was approximately 10(-5) M. At higher concentrations (5 x 10(-4) M) migration was not induced or was inhibited. When tested by subcutaneous implant assays in rabbits, cis-4-hydroxy-L-proline and 2-azetidinecarboxylic acid consistently elicited a marked angiogenic response. Whereas these compounds are known to modulate collagen synthesis and secretion, the concentrations at which they are effective inducers of migration suggest that they may have a more specific target than general collagen synthesis.     

Retinal capillary endothelial cells prefer different substrates for growth and migration

Recent studies have shown that the extracellular matrix modifies the behaviour of endothelial cells. We have studied the effects of extracellular matrix components on retinal capillary endothelial cell migration and proliferation. Bovine retinal capillary endothelial cells were selectively cultured from collagenase-digested microvessel fragments. In a filter system for the assessment of migration, endothelial cells responded to substrate-bound fibronectin but not to soluble fibronectin. Cell migration on collagen- or gelatin-coated filters was minimal, and these cells failed to adopt a spread morphology, remaining instead as round cells. Cell replication was quantified using a protein dye binding assay for adherent cells in 96 well plates. Serum was essential for growth irrespective of the substrate. Cells harvested from microvessel cultures proliferated more rapidly on collagen- and gelatin-coated plastic than on fibronectin and were unaffected by additions to the medium such as endothelial cell conditioned medium, whereas cells proliferating directly from the microvessels grew at a faster rate on fibronectin and also responded to conditioned medium supplement. When cultured on collagen gels, initial microvessel cells and harvested cells required surface fibronectin in order to adopt a cobblestone morphology. These results show that fibronectin is a requirement for bovine retinal capillary endothelial cell migration, but proliferation of these cells can be supported, with slight differences, by both fibronectin and collagen provided serum growth factors are present. These findings are relevant to the early phase of angiogenesis in which migration and proliferation of endothelial cells occurs.     

Hypothesis: the target cell of GM-CSF is a macrophage precursor capable to produce cells with the property to secrete a G-CSF like activity.

The induction of granulocyte and macrophage colony formation by the granulocyte-macrophage colony stimulating factor (GM-CSF) on bone marrow cells (BMC) was evaluated as a function of time in agar cultures. We found that while macrophage cell clusters were very abundant on the first two days of culture, granulocytic cell clusters did not appear until the third day. We also found that macrophage colonies were present from the fourth day of culture, while granulocyte colonies did not appear until the fifth day. When two day cell clusters were transferred to cultures with GM-CSF we observed that only macrophage-colonies developed. On the other hand, when four day clusters were transferred, both granulocyte and macrophage colony formation was obtained in a similar way as the one obtained when using GM-CSF with fresh BMC. Two day clusters did not respond to granulocyte colony stimulating factor (G-CSF) while fourth day clusters generated granulocytic colonies in a similar way as when G-CSF was used with fresh BMC. In order to test the hypothesis that granulocyte colony formation in these assays could be a result of the secretion of G-CSF by the macrophages previously induced by GM-CSF, lysates from macrophage colonies were used to induce colony formation on BMC. We observed that colonies, mainly granulocytic, were induced in a similar way as when G-CSF was used. Finally, the possibility that GM-CSF is just a macrophage inducer with the property to produce cells that secrete G-CSF is discussed.     

Improved single-strand DNA sizing accuracy in capillary electrophoresis.

Interpolation algorithms can be developed to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragments. We used the anomalous migration of ssDNA fragments to optimize denaturation conditions for capillary electrophoresis. The capillary electrophoretic system uses a refillable polymer that both coats the capillary wall to suppress electro-osmotic flow and acts as the sieving matrix. The addition of 8 M urea to the polymer solution, as in slab gel electrophoresis, is insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. The sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 degrees C). the effect of these two denaturing strategies is additive, and the best accuracy and precision in sizing results are obtained with a combination of chemical and thermal denaturation.     

Modulation of lymphocyte motility by macrophages

Motility of lymphocytes plays a significant role in their functions. Because macrophages frequently associate with lymphocytes in lymphoid tissues and inflammatory sites, they are likely to be important in regulating lymphocyte motility. In this study, we identified a chemokinetic activity in macrophage culture supernatants. Interestingly, this activity could be detected by the capillary migration assay but not by the more commonly used Boyden chamber chemotaxis assay. Colchicine, on the other hand, was chemokinetic for lymphocytes in the Boyden chamber chemotaxis assay but not in the capillary migration assay. Both these observations and previous studies on the morphology of motile lymphocytes on two-dimensional (2-D) surfaces (capillary migration assay) and in 3-D matrices (Boyden chamber chemotaxis assay) suggest that lymphocytes possess more than one motility mechanism--one for 2-D surfaces and one for 3-D matrices. We propose that the macrophage-derived chemokinetic activity described herein only affected the motility mechanism on 2-D surfaces. In addition, we also observed that the chemokinetic activity was produced by "resting" macrophages and could not be augmented by further activation. Finally, the effect was greatest on mature T cells. We propose that this factor plays an important role in facilitating cell interactions within lymphoid tissues and inflammatory sites.     

Saikosaponin C induces endothelial cells growth, migration and capillary tube formation

Saikosaponin C is one of the saikosaponins that are consisted in a Chinese herb, Radix Bupleuri. Recently, saikosaponins have been reported to have properties of cell growth inhibition, inducing cancer cells differentiation and apoptosis. However, saikosaponin C had no correlation with cell growth inhibition. In this study, we investigated the role of saikosaponin C on the growth of endothelial cells and angiogenesis. We found that saikosaponin C yielded a potent effect on inducing human umbilical vein endothelial cells (HUVECs) viability and growth. In addition to inducing endothelial cells growth, saikosaponin C also induced endothelial cells migration and capillary tube formation. The gene expression or activation of matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF) and the p42/p44 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells growth, migration and angiogenesis were also induced by saikosaponin C. From these results, we suggest that saikosaponin C may have the potential for therapeutic angiogenesis but is not suitable for cancer therapy.     

Comparative migration of guinea pig T and B lymphocytes from capillary tubes

Using a capillary tube migration technique, a comparison was made between the random mobility of separated guinea pig T- and B-lymph node lymphocyte subpopulations, and macrophage-rich peritoneal exudate cells. A decrease in random movement was found in that order, which would fit with the hypothesis that migration on a substrate is an adherence-dependent phenomenon. In order to characterize a possible dichotomy between T-and B-cell locomotion, the effects of several factors which might interfere with their migration were studied. These factors included drugs which affect cell metabolism, cell surface ligands, and some factors which may play a role in inflammatory foci (acidity, immune complexes, and lymphokines). The results emphasize the similarity in the mode of locomotion of T and B lymphocytes. However, a remarkable difference was found in the stronger inhibition of B-cell migration by pH values below pH 7.0. The relevance of these findings to the migration of T and B lymphocytes into inflammatory foci is discussed.     

Cyclooxygenase-2-derived prostaglandin D(2) is an early anti-inflammatory signal in experimental colitis

The ability of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors to exacerbate inflammatory bowel disease suggests that prostaglandins are important anti-inflammatory mediators in this context. Prostaglandin D(2) has been suggested to exert anti-inflammatory effects. We investigated the possibility that prostaglandin D(2) derived from cyclooxygenase-2 plays an important role in downregulating colonic inflammation in rats. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various times thereafter (from 1 h to 7 days), colonic prostaglandin synthesis and myeloperoxidase activity (index of granulocyte infiltration) were measured. Prostaglandin D(2) synthesis was elevated >4-fold above controls within 1-3 h of induction of colitis, preceding significant granulocyte infiltration. Treatment with a selective cyclooxygenase-2 inhibitor abolished the increase in prostaglandin D(2) synthesis and caused a doubling of granulocyte infiltration. Colonic granulocyte infiltration was significantly reduced by administration of prostaglandin D(2) or a DP receptor agonist (BW-245C). These results demonstrate that induction of colitis results in a rapid increase in prostaglandin D(2) synthesis via cyclooxygenase-2. Prostaglandin D(2) downregulates granulocyte infiltration into the colonic mucosa, probably through the DP receptor.     

Guided migration as a novel mechanism of capillary network remodeling is regulated by basic fibroblast growth factor

 To investigate mechanisms of capillary network remodeling, we developed a serum-free angiogenesis in vitro system in three-dimensional fibrin matrices which allows the study of directional growth of endothelial sprouts, anastomosis, and remodeling (’pruning’) of the primitive plexus toward more elaborated capillary trees. To follow the movements of living endothelial cells by inverse-fluorescence microscopy, we cocultured unlabeled endothelial cells with endothelial cells labeled with the carbocyanine dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). We show that elongation and retraction of neighboring capillary sprouts occurs simultaneously, resembling a tug-of-war by which endothelial cells are withdrawn from shortening sprouts to become incorporated in other sprouts nearby. For the first time, we directly demonstrate the long-suspected parallel sliding movement of endothelial cells. We show that cell migration persists within immature capillaries even after sprouts have merged to continuous capillary loops, leading to overlapping growth of opposing sprout tips. As a novel concept of capillary remodeling, we distinguish two types of endothelial cell migration: sprouting and guided migration. Sprouting is the de novo invasion of a matrix by endothelial cells, and guided migration is the locomotion of cells along preexistent capillary-like structures. We show that guided migration leads to remodeling of immature capillary networks and to the retraction of sprouts. We describe a method for quantification of sprouting versus guided migration in DiI-mosaic-labeled capillary networks, and we present evidence that endothelial cell-derived basic fibroblast growth factor serves as a chemotactic signal for other cells to migrate along a preestablished capillary-like structure. Accepted: 3 November 1997     

Dynamic coating for fast and reproducible determination of basic drugs by capillary electrophoresis with diode-array detection and mass spectrometry

The double coating principle of CEofix buffers was evaluated for the analysis of some basic drugs by capillary electrophoresis-diode-array detection (CE-DAD) and capillary electrophoresis-mass spectrometry (CE-MS). The involatile phosphate present in original low pH CEofix, was replaced with formic acid for hyphenation of CE with MS. The double coating produces a substantial and highly reproducible electroosmotic flow (EOF), even at low pH. The rinsing procedure and electrolyte composition were optimized for both CE-DAD and CE-MS. The system was evaluated with the analysis of a mixture of basic drugs and a spiked urine sample enriched by solid-phase extraction (SPE). The R.S.D. values on the migration time and peak area measured for 28 analyses with CE-DAD were below 0.25 and 2.40%, respectively. For CE-MS, the R.S.D. on the migration time was 0.85% or less and the area precision ranged from 5.65 to 14.33% (for seven injections). The LOD with the developed CE-MS method was below 50 ppb for all five drug standards tested.     

N-glycan biosynthesis inhibitors induce in vitro anticancer activity in colorectal cancer cells

During malignant transformation, changes in the expression profile of glycans may be involved in a variety of events, including the loss of cell-cell and cell-matrix adhesion, migration, invasion, and evasion of apoptosis. Therefore, modulation of glycan expression with drugs has promising therapeutic potential for various cancer types. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors (swainsonine and tunicamycin) in cells derived from colorectal cancer (CRC). We also examined whether these inhibitors are able to induce radiosensitization and toxicity when used in combination with cisplatin or irinotecan, two current anticancer drugs. Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in undifferentiated HCT-116 colon cancer cells, whereas swainsonine only inhibits cell migration. We also observed that tunicamycin, but not swainsonine, caused radiosensitivity in HCT-116 cells. Moreover, the combination of swainsonine with cisplatin or irinotecan enhanced their toxicity in HCT-116 cells, while the combination of tunicamycin with these drugs had no effect. Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for CRC treatment because inhibition of this process induced anticancer activity in vitro. Additionally, the inhibition of the N-glycan biosynthesis in combination with chemotherapic drugs is a promising therapeutic strategy for enhancing radiation therapy.     

The effect of platinum derivatives on interfacial properties of DNA

The interaction of DNA modified by the binding of various platinum compounds with an electrically charged mercury surface was studied by means of linear sweep voltammetry. It was found that DNA and its adducts with antitumour active cis-diammine-dichloroplatinum(II) (cis-DDP) on the one hand and antitumour inactive trans-diamminedichloroplatinum(II) (trans-DDP) and diethylenetriaminechloroplatinum(II) chloride (dien-Pt) on the other were unwound due to their adsorption on the negatively charged mercury surface polarized to the potentials of a narrow region around -1.2 V (vs. saturated calomel electrode). The modification of DNA by bifunctional platinum compounds (cis- and trans-DDP) resulted in a substantial lowering of the extent of this interfacial conformational rearrangement, the modification by trans-DDP being more effective. The modification of DNA by monofunctional dien-Pt influenced the unwinding of DNA on the mercury surface only negligibly. It has been concluded that in particular interstrand cross-links induced by platinum compounds in DNA are responsible for the effect of these drugs on the extent of the interfacial unwinding of DNA. This conclusion is in good agreement with the view that among the lesions induced in DNA by platinum compounds, the interstrand cross-links are of less significance from the point of view of the antitumour efficacy of these inorganic drugs.     

Heat Shock Protein 27 Plays Two Distinct Roles in Controlling Human Breast Cancer Cell Migration on Laminin-5

It has recently been reported that phosphorylation of the small heat shock protein 27 (hsp27) enhances p38 MAP kinase dependent migration of bovine and human vascular endothelial cells. We have examined the role of hsp27 in controlling the constitutive migration of human breast cancer cells on the extracellular matrix molecule laminin-5. In a haptotaxis assay, anisomycin- or heat shock-induced phosphorylation of hsp27 enhances migration of MDA-MB-231 breast cancer cells constitutively overexpressing hsp27. Under these conditions, hsp27 redistributes to the nucleus. Unphosphorylated hsp27, which remains in the cytosol, induces resistance to a subset of drugs that inhibit haptotactic migration of these cells. We conclude that hsp27 plays two distinct roles in controlling migration of breast cancer cells: phosphorylated hsp27 enhances migration, while unphosphorylated hsp27 can sustain migration in the presence of inhibitory drugs.     

Water extracts ofAralia elata root bark enhances migration and matrix metalloproteinases secretion in porcine coronary artery endothelial cells

Aralia elata is an edible mountain vegetable. Angiogenesis, the formation of new blood vessels, is a process involving migration, proliferation and cell differentiation, as well as the formation of new capillary structures. Matrix metalloproteinases (MMPs) plays an important role in angiogenesis. The development of a functional vascular system requires a variety of growth factors, their receptors, and intracellular signals. This study examines the effects of water extracts from: (i)A. elata root bark (Aralia extracts); (ii) a combination of Aralia extracts and fibroblast growth factors (FGF-2) on cultured porcine coronary artery endothelial cells (PCAECs). Aralia extracts induced the migration of PCAECs, which was inhibited by MMPs inhibitors. Combining Aralia extracts and FGF-2 enhanced the migration and the secretion of MMP-2 and MMP-9 from PCAECs. We postulated that the Aralia extracts, which induced migrating activity in PCAECs, may be accomplished by increased secretion levels of MMP-2 and MMP-9.