Distribution of antioxidant enzymes in developing human lung, respiratory distress syndrome, and bronchopulmonary dysplasia.

We studied cell-specific protein expression of all the major antioxidant enzymes (AOEs) and related proteins, such as copper-zinc superoxide dismutase (CuZnSOD), manganese SOD (MnSOD), extracellular SOD (ECSOD), catalase, the heavy and light chains of gamma-glutamylcysteine synthetase (gamma-GCS-l and gamma-GCS-h, also called glutamate cysteine ligase), the rate-limiting enzyme in glutathione synthesis, hemeoxygenase-1 (HO-1), and thioredoxin (Trx), in developing human lung, respiratory distress syndrome, and bronchopulmonary dysplasia by immunohistochemistry. Generally, after 17 weeks of gestational age, MnSOD was predominantly expressed in bronchial epithelium, alveolar epithelium, and macrophages, CuZnSOD was expressed in bronchial epithelium, ECSOD was expressed in bronchial epithelium, vascular endothelium, and the extracellular matrix, catalase was expressed in bronchial epithelium and alveolar macrophages, gamma-GCS-h was expressed in bronchial epithelium and endothelium, and gamma-GCS-l was expressed in bronchial epithelium. Trx was restricted to bronchial epithelium and to a lesser extent to alveolar macrophages, and HO-1 found in alveolar macrophages. Basically, the expression of these enzymes was similar in normal and diseased lung. It can be concluded that various AOEs and related proteins differ in their distribution and expression in lung before term, but generally it seems that infants are better adapted to high oxygen tension than might be expected.     

Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.     

Tissue distribution of the death ligand TRAIL and its receptors.

Recombinant human (rh) TNF-related apoptosis-inducing ligand (TRAIL) harbors potential as an anticancer agent. RhTRAIL induces apoptosis via the TRAIL receptors TRAIL-R1 and TRAIL-R2 in tumors and is non-toxic to nonhuman primates. Because limited data are available about TRAIL receptor distribution, we performed an immunohistochemical (IHC) analysis of the expression of TRAIL-R1, TRAIL-R2, the anti-apoptotic TRAIL receptor TRAIL-R3, and TRAIL in normal human and chimpanzee tissues. In humans, hepatocytes stained positive for TRAIL and TRAIL receptors and bile duct epithelium for TRAIL, TRAIL-R1, and TRAIL-R3. In brains, neurons expressed TRAIL-R1, TRAIL-R2, TRAIL-R3 but no TRAIL. In kidneys, TRAIL-R3 was negative, tubuli contorti expressed TRAIL-R1, TRAIL-R2, and TRAIL, and cells in Henle's loop expressed only TRAIL-R2. Heart myocytes showed positivity for all proteins studied. In colon, TRAIL-R1, TRAIL-R2, and TRAIL were present. Germ and Leydig cells were positive for all proteins studied. Endothelium in liver, heart, kidney, and testis lacked TRAIL-R1 and TRAIL-R2. In alveolar septa and bronchial epithelium TRAIL-R2 was expressed, brain vascular endothelium expressed TRAIL-R2 and TRAIL-R3, and in heart vascular endothelium only TRAIL-R3 was present. Only a few differences were observed between human and chimpanzee liver, brain, and kidney. In contrast to human, chimpanzee bile duct epithelium lacked TRAIL, TRAIL-R1, and TRAIL-R3, lung and colon showed no TRAIL or its receptors, TRAIL-R3 was absent in germ and Leydig cells, and vascular endothelium showed only TRAIL-R2 expression in the brain. In conclusion, comparable expression of TRAIL and TRAIL receptors was observed in human and chimpanzee tissues. Lack of liver toxicity in chimpanzees after rhTRAIL administration despite TRAIL-R1 and TRAIL-R2 expression is reassuring for rhTRAIL application in humans.     

Regulated production of the chemokine CCL28 in human colon epithelium

The chemokine CCL28 is constitutively expressed by epithelial cells at several mucosal sites and is thought to function as a homeostatic chemoattractant of subpopulations of T cells and IgA B cells and to mediate antimicrobial activity. We report herein on the regulation of CCL28 in human colon epithelium by the proinflammatory cytokine IL-1, bacterial flagellin, and n-butyrate, a product of microbial metabolism. In vivo, CCL28 was markedly increased in the epithelium of pathologically inflamed compared with normal human colon. Human colon and small intestinal xenografts were used to model human intestinal epithelium in vivo. Xenografts constitutively expressed little, if any, CCL28 mRNA or protein. After stimulation with the proinflammatory cytokine IL-1, CCL28 mRNA and protein were significantly increased in the epithelium of colon but not small intestinal xenografts, although both upregulated the expression of another prototypic chemokine, CXCL8, in response to the identical stimulus. In studies of CCL28 regulation using human colon epithelial cell lines, proinflammatory stimuli, including IL-1, bacterial flagellin, and bacterial infection, significantly upregulated CCL28 mRNA expression and protein production. In addition, CCL28 mRNA expression and protein secretion by those cells were significantly increased by the short-chain fatty acid n-butyrate, and IL-1- or flagellin-stimulated upregulation of CCL28 by colon epithelial cells was synergistically increased by pretreatment of cells with n-butyrate. Consistent with its upregulated expression by proinflammatory stimuli, CCL28 mRNA expression was attenuated by pharmacological inhibitors of NF-kappaB activation. These findings indicate that CCL28 functions as an "inflammatory" chemokine in human colon epithelium and suggest the notion that CCL28 may act to counterregulate colonic inflammation.     

Expression of caveolin-1 and podocalyxin in rat lungs challenged with 2-kDa macrophage-activating lipopeptide and Flt3L

Caveolin-1 is one of the important regulators of vascular permeability in inflamed lungs. Podocalyxin is a CD34 protein expressed on vascular endothelium and has a role in podocyte development in the kidney. Few data are available on the expression of caveolin-1 and podocalyxin in lungs challenged with Toll-like receptor 2 (TLR2) agonists such as mycoplasma-derived macrophage activating lipopeptide or with immune modulators such as Fms-like tyrosine kinase receptor-3 ligand (Flt3L), which expands dendritic cell populations in the lung. Because of the significance of pathogen-derived molecules that act through TLR2 and of the role of immune modulators in lung physiology, we examine the immunohistochemical expression of caveolin-1 and podocalyxin in lungs from rats challenged with a 2-kDa macrophage-activating lipopeptide (MALP-2) and Flt3L. Normal rat lungs expressed caveolin-1 in alveolar septa, vascular endothelium and airway epithelium, especially along the lateral borders of epithelial cells but not in alveolar macrophages. MALP-2 and Flt3L decreased and increased, respectively, the expression of caveolin-1. Caveolin-1 expression seemed to increase in microvessels in bronchiole-associated lymphoid tissue (BALT) in Flt3L-challenged lungs but not in normal or MALP-2-treated lungs. Podocalyxin was absent in the epithelium and alveolar macrophages but was present in the vasculature of control, Flt3L- and MALP-2-treated rats. Compared with control and MALP-2-treated rats, Flt3L-treated lungs showed greater expression of podocalyxin in BALT vasculature and at the interface of monocytes and the endothelium. These immunohistochemical data describing the altered expression of caveolin-1 and podocalyxin in lungs treated with MALP-2 or Flt3L encourage further mechanistic studies on the role of podocalyxin and caveolin-1 in lung inflammation.     

Light microscopic immunohistochemical analysis of the distribution of group II phospholipase A2 in human digestive organs.

The light microscopic and immunohistochemical distribution of human Group II phospholipase A2 (M-PLA2) in digestive organs of both human fetus and adult, with a new monoclonal antibody (MAb) against M-PLA2, was investigated semiquantitatively. The immunoreactivity was distributed similarly in the adult and fetal epithelium of the esophagus, duodenum, and small intestine, and in the acinar, islet, and duct cells of the pancreas. The epithelium of adult gallbladder was immunoreactive. Paneth cells, especially the secretory apparatus, were strongly immunoreactive. Hepatic Küpffer cells and macrophages of the adult spleen were also immunoreactive. These results suggest that these cells contain secretory-type Group II PLA2, which may be involved in host defensive mechanisms, such as phagocytosis in human digestive organs. In the adult colon, the immunoreactivity was observed only in the ascending colon and was not found in the transverse, descending, sigmoid, or rectal colon. The immunoreactivity was not found in fetal colon. Similarly, immunoreactivity was found in hepatocytes and Küpffer cells of adult liver but not in fetal liver. By contrast, strong immunoreactivity was observed in the epithelium of the fetal stomach but not in adult stomach except in gastric neck cells. This suggests that the expression of M-PLA2 may be related to cell differentiation in particular organs.     

Cellular and subcellular localization of paralemmin-1, a protein involved in cell shape control,in the rat brain,adrenal gland and kidney

Paralemmin-1 is a phosphoprotein, lipid-anchored to the cytoplasmic face of membranes and implicated in plasma membrane dynamics and cell process formation. We report an immunoperoxidase histochemical analysis of the cellular and subcellular localization of paralemmin-1 in the rat tissues where its expression is highest: the brain, the adrenal gland and the kidney. Paralemmin-1 is detected throughout the brain, in neuronal perikarya, axons and dendrites including dendritic spines and also in glial processes. In the adrenal gland, paralemmin-1 is highly expressed in the medulla. The kidney displays a pattern of differential paralemmin-1 expression in various structures and cell types, with high concentrations in cells of the parietal epithelium of Bowman’s capsule, intermediate tubules, distal tubules and principal cells of outer medullary collecting ducts. Mosaics of paralemmin-positive and paralemmin-negative cells are observed in proximal tubules, the parietal epithelium of Bowman’s capsule and the endothelium of many blood vessels. Plasma membrane association in epithelia is often polarized: paralemmin-1 concentrates at the apical membranes of adrenal chromaffin cells, but at the basolateral plasma membranes of proximal and distal tubule cells in the kidney. Paralemmin-1 immunoreactivity exhibits a spotted pattern and can be seen both at plasma membranes and within the cytoplasm, where it is often associated with endomembranes. This discontinuous distribution and the detergent extraction properties of paralemmin-1 suggest an association with lipid microdomains. The findings are consistent with a role for paralemmin-1 in the formation and stabilization of plasma membrane elaborations, in neurons as well as in other cell types.     

The laxative effect of bisacodyl is attributable to decreased aquaporin-3 expression in the colon induced by increased PGE2 secretion from macrophages

The purpose of this study was to investigate the role of aquaporin3 (AQP3) in the colon in the laxative effect of bisacodyl. After oral administration of bisacodyl to rats, AQP3, macrophages, cyclooxygenase 2 (COX2), and prostaglandin E(2) (PGE(2)) were examined in the colon. The mechanism by which bisacodyl decreases the expression of AQP3 was examined using HT-29 and Raw264.7 cells. When diarrhea occurred, a significant increase in the expression of PGE(2) and a decrease in AQP3 expression were observed. Immunostaining showed COX2 expression only in macrophages. The PGE(2) concentration increased significantly 30 min after the addition of bisacodyl to Raw264.7 cells. Thirty minutes after PGE(2) addition to HT-29 cells, the AQP3 expression level decreased to 40% of the control. When pretreated with indomethacin, bisacodyl did not induce an increase in the colon PGE(2) level, a decrease in the AQP3 expression level, or diarrhea. The results suggest that bisacodyl may decrease the expression of AQP3 in the colon, which inhibits water transfer from the luminal to the vascular side and leads to a laxative effect. This study also showed that direct activation of colon macrophages by bisacodyl increases the secretion of PGE(2), which acts as a paracrine factor and decreases AQP3 expression in colon mucosal epithelial cells.     

Production of insulin-like growth factor binding-proteins by bovine adrenomedullary cells: differential regulation by IGF-I and dexamethasone

In the present study we examined the production of insulin-like growth factor binding proteins (IGFBPs), in chromaffin cells, a model system for sympathetic neurons. Four IGFBPs of approximately 27, approximately 31, approximately 36 and a doublet of approximately 45-50 kDa, detected in Western ligand blots of conditioned medium, were identified in Western immunoblots as IGFBP-4, IGFBP-5, IGFBP-2 and IGFBP-3, respectively. In ligand blots IGFBP-3 and IGFBP-4 appeared as the most prominent species. IGF-I (1 nM) enhanced release of IGFBP-3 while dexamethasone (1 nM) diminished release of IGFBP-4. No significant proteolytic degradation of the IGFBPs was demonstrated. Cycloheximide completely attenuated release of the IGFBPs, indicating dependency on new synthesis of the proteins. These findings are consistent with autocrine modulation of the IGF system in bovine adrenomedullary chromaffin cells by IGFBPs. Furthermore, the specific stimulatory and inhibitory effects of IGF-I and dexamethasone, respectively, on release of the predominant species of IGFBP-3 and IGFBP-4, suggested that IGFBP production may be selectively modulated in a positive and negative manner.     

Expression of adhesion molecules in lungs of mice infected with Paracoccidioides brasiliensis conidia

In infected tissues, leukocyte recruitment is mediated by interactions between adhesion molecules, expressed on activated vascular endothelial cells, and ligands present on circulating cells. We evaluated the inflammatory response and the expression of cellular adhesion molecules (ICAM-1, VCAM-1, CD18, LFA-1 and Mac-1) in lungs of BALB/c mice infected with Paracoccidioides brasiliensis conidia. When compared with uninfected animals, infected mice had a significant increase in the inflammatory response during the first 4 days, peaking 2-3 days post-challenge, 40.3% vs. 0.0% and 41.8% vs. 0.7%, respectively. This inflammatory infiltrate was composed mainly of neutrophils and macrophages with a few eosinophils and lymphocytes. An increase in the intensity of immunofluorescence (IF) for ICAM-1 was also observed during days 1-4. ICAM-1 was present in bronchiolar epithelium, type II pneumocytes, and macrophages, as well as on vascular endothelium. The control animals presented ICAM-1 constitutively. In infected mice, VCAM-1 was only observed on vascular endothelium during the first 2 days, with some macrophages expressing this molecule throughout the study periods. CD18 and Mac-1 but not LFA-1 were expressed with a high intensity on neutrophils and macrophages present in the inflammatory infiltrate. In addition, we observed a significant decrease in Colony forming units (CFUs) after the first 2 days post-challenge. These findings suggest that during these early stages, up-regulation of ICAM-1, VCAM-1, CD18 and Mac-1 expression occurs, participating in the inflammatory process and as such, in the pathogenesis of paracoccidioidomycosis (PCM).     

LacZ expression in Fut2-LacZ reporter mice reveals estrogen-regulated endocervical glandular expression during estrous cycle, hormone replacement, and pregnancy

The secretor gene (FUT2) encodes an alpha(1,2)fucosyltransferase (E.C. 2.4.1.69) that elaborates alpha(1,2)fucose residues on mucosal epithelium and secreted mucins. Though uterine alpha(1,2)fucosylated glycans have been proposed to be involved in embryo adhesion, mice with a homozygous null mutation of Fut2 displayed normal fertility. To help develop alternative hypotheses for function, the cell type and regulation of Fut2 expression during the estrous cycle, hormone replacement, and pregnancy was examined in Fut2-LacZ reporter mice containing targeted replacement of Fut2 with bacterial lacZ. LacZ expression in the reproductive tract of Fut2-LacZ mice is most prominent in the glandular epithelium of the endocervix during estrus and pregnancy. Nuclear LacZ expression identifies cell-specific expression of Fut2 in mucus-secreting cells of the endocervix, uterine glands, foveolar pit and chief cells of the stomach, and goblet cells of the colon. In ovariectomized Fut2-LacZ mice, estradiol treatment stimulates X-gal staining in endocervix and uterus but does not affect expression in stomach and colon. Northern blot analysis in wild-type mice shows 15-fold elevations of Fut2 steady-state mRNA with estradiol treatment, whereas Fut1 varies little. Fut2 levels in the glandular stomach and distal colon remain constant, and uterine Fut2 levels vary eightfold during the estrous cycle. These data represent the first demonstration of a glycosyltransferase gene under tissue-specific hormonal regulation in a LacZ reporter mouse model. Endocervical expression of Fut2 in estrus and pregnancy may modify cervical mucus barrier properties from microbial infection analogous to the potential role of mucosal glycans in humans.     

Functional mitochondria are required for O2 but not CO2 sensing in immortalized adrenomedullary chromaffin cells

Catecholamine (CAT) release from adrenomedullary chromaffin cells (AMC) in response to stressors such as low O(2) (hypoxia) and elevated CO(2)/H(+) is critical during adaptation of the newborn to extrauterine life. Using a surrogate model based on a v-myc immortalized adrenal chromaffin cell line (i.e., MAH cells), combined with genetic perturbation of mitochondrial function, we tested the hypothesis that functional mitochondria are required for O(2) sensing. Wild-type MAH cells responded to both hypoxia and increased CO(2) (hypercapnia) with K(+) current inhibition and membrane depolarization. Additionally, these stimuli caused a rise in cytosolic Ca(2+) and CAT secretion, determined by fura-2 spectrofluorimetry and carbon fiber amperometry, respectively. In contrast, mitochondria-deficient (rho(0)) MAH cells were hypoxia insensitive, although responses to hypercapnia and expression of several markers, including carbonic anhydrase II, remained intact. Rotenone (1 microM), a mitochondrial complex I blocker known to mimic and occlude the effects of hypoxia in primary AMC, was effective in wild-type but not rho(0) MAH cells. These data demonstrate that functional mitochondria are involved in hypoxia-sensing by adrenal chromaffin cells. We also show for the first time that, like their neonatal chromaffin cell counterparts, MAH cells are CO(2) sensors; however, this property is independent of functional mitochondria.     

Regulated MIP-3alpha/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity

Human intestinal epithelial cells secrete an array of chemokines known to signal the trafficking of neutrophils and monocytes important in innate mucosal immunity. We hypothesized that intestinal epithelium may also have the capacity to play a role in signaling host adaptive immunity. The CC chemokine macrophage inflammatory protein (MIP)-3alpha/CCL20 is chemotactic for immature dendritic cells and CD45RO(+) T cells that are important components of the host adaptive immune system. In these studies, we demonstrate the widespread production and regulated expression of MIP-3alpha by human intestinal epithelium. Several intestinal epithelial cell lines were shown to constitutively express MIP-3alpha mRNA. Moreover, MIP-3alpha mRNA expression and protein production were upregulated by stimulation of intestinal epithelial cells with the proinflammatory cytokines tumor necrosis factor-alpha or interleukin-1alpha or in response to infection with the enteric bacterial pathogens Salmonella or enteroinvasive Escherichia coli. In addition, MIP-3alpha was shown to function as a nuclear factor-kappaB target gene. In vitro findings were paralleled in vivo by increased expression of MIP-3alpha in the epithelium of cytokine-stimulated or bacteria-infected human intestinal xenografts and in the epithelium of inflamed human colon. Mucosal T cells, other mucosal mononuclear cells, and intestinal epithelial cells expressed CCR6, the cognate receptor for MIP-3alpha. The constitutive and regulated expression of MIP-3alpha by human intestinal epithelium is consistent with a role for epithelial cell-produced MIP-3alpha in modulating mucosal adaptive immune responses.     

Variations in adrenal gland medulla and dopamine effects induced by the lack of Irs2

The adrenomedullary chromaffin cells’ hormonal pathway has been related to the pathophysiology of diabetes mellitus. In mice, the deletion of insulin receptor substrate type 2 (Irs2) causes peripheral insulin resistance and reduction in β-cell mass, leading to overt diabetes, with gender differences on adrenergic signaling. To further unravel the relevance of Irs2 on glycemic control, we analyzed in adult Irs2 deficient (Irs2^?/?) mice, of both sexes but still normoglycemic, dopamine effects on insulin secretion and glycerol release, as well as their adrenal medulla by an immunohistochemical and morphologic approach. In isolated islets, 10 μM dopamine significantly inhibited insulin release in wild-type (WT) and female Irs2^?/? mice; however, male Irs2^?/? islets were insensitive to that catecholamine. Similarly, on isolated adipocytes, gender differences were observed between WT and Irs2^?/? mice in basal and evoked glycerol release with crescent concentrations of dopamine. By immunohistochemistry, reactivity to tyrosine hydroxylase (TH) in female mice was significantly higher in the adrenal medulla of Irs2^?/? compared to WT; although no differences for TH-immunopositivity were observed between the male groups of mice. However, compared to their corresponding WT animals, adrenomedullary chromaffin cells of Irs2^?/? mice showed a significant decrease in the cellular and nuclear areas, and even in their percentage of apoptosis. Therefore, our observations suggest that, together with gender differences on dopamine responses in Irs2^?/? mice, disturbances in adrenomedullary chromaffin cells could be related to deficiency of Irs2. Accordingly, Irs2 could be necessary for adequate glucose homeostasis and maintenance of the population of the adrenomedullary chromaffin cells.     

Reduction of endogenous TGF-beta does not affect phenotypic development of sympathoadrenal progenitors into adrenal chromaffin cells.

Adrenal chromaffin cells and sympathetic neurons are related, but phenotypically distinct derivatives of the neural crest. Molecular cues that determine the chromaffin cell phenotype have not yet been identified; in contrast to a widely held belief, glucocorticoid signaling is apparently not relevant (Development 126 (1999) 2935). Transforming growth factor-betas (TGF-betas) regulate various aspects of embryonic development and are expressed in the environment of sympathoadrenal (SA) progenitor cells. We have previously shown that neutralization of endogenous TGF-beta from E4 to E8 in the quail embryo significantly increases numbers of adrenal tyrosine hydroxylase-positive cells. Whether endogenous TGF-beta may also be involved in influencing phenotypic development of adrenal chromaffin cells and their SA progenitors has not been analyzed. We now demonstrate that neutralization of endogenous TGF-beta1, -beta2 and -beta3 with a pan-anti-TGF-beta antibody in quail embryos during distinct time windows does not alter phenotypic development of chromaffin cells. In situ hybridizations revealed unaltered expression of neurofilament (NF-160), synaptotagmin I and neurexin I in adrenal glands. Likewise, the NF-associated antigen 3A10, and polyphosphorylated NF epitopes (RT 97) were unaltered. Most importantly, the typical ultrastructure of adrenal chromaffin cells including their large chromaffin secretory granules, a hallmark of the neuroendocrine phenotype, which distinguishes them from sympathetic neurons, was not affected. We therefore conclude that neutralization of endogenous TGF-beta influences chromaffin cell proliferation, but does not interfere with the development of the typical chromaffin cell phenotype.     

Role of ascorbic acid in dopamine beta-hydroxylation. The endogenous enzyme cofactor and putative electron donor for cofactor regeneration

The role(s) of ascorbic acid in dopamine beta-hydroxylation was studied in primary cultures of bovine adrenomedullary chromaffin cells and in isolated bovine adrenomedullary chromaffin vesicles. Dopamine beta-hydroxylase activity was assessed by measuring the rate of conversion of tyramine to octopamine. The ascorbic acid content of chromaffin cells declined with time in culture and the dopamine beta-hydroxylase activity of ascorbate-depleted cells was low. Ascorbate additions to ascorbate-depleted cells increased both the intracellular ascorbate concentrations and the rates of dopamine beta-hydroxylation. Ascorbate uptake into the cells was rapid; however, the onset of enhanced octopamine synthesis by added ascorbate was delayed by several hours and closely followed the time course for accumulation of the newly taken up ascorbate into the chromaffin vesicle. The amount of octopamine synthesized by the chromaffin cells exceeded the intracellular ascorbate content and ascorbate levels were maintained during dopamine beta-hydroxylation in the absence of external ascorbate. This suggests an efficient recycling of ascorbate. In contrast to intact cells, ascorbic acid was depleted during octopamine synthesis in isolated chromaffin vesicles. The molar ratio of octopamine formed to ascorbate depleted was close to unity. Thus, the recycling of intravesicular ascorbate depends on an extravesicular factor(s). The depletion of intravesicular ascorbate during dopamine beta-hydroxylation was prevented by the addition of nonpermeant extravesicular electron donors such as ascorbate or glucoascorbate. This suggests that intravesicular ascorbate is maintained in the reduced state by electron transport across the vesicle membrane. These results are compatible with the hypothesis that both intra- and extravesicular ascorbate participate in the regulation of dopamine beta-hydroxylase. Intravesicular ascorbate is the cofactor for the enzyme. Cytosolic ascorbate is most likely the electron donor for the vesicle-membrane electron transport system which maintains the intravesicular cofactor concentration.     

Leukotriene B4 receptors: novel roles in immunological regulations

Mammals have at least two receptors for LTB4; high-affinity BLT1 and low-affinity BLT2, both of which are GPCRs. 12-HHT serves as a more potent and abundant ligand for BLT2 than LTB4. BLT1 is expressed in a variety of inflammatory and immune cells including granulocytes, eosinophils, macrophages, differentiated Th1, Th2 and Th17 cells, effecter CD8+ T cells, dendritic cells and osteoclasts. BLT1 antagonists will be beneficial for the treatment of various diseases such as bronchial asthma, multiple sclerosis, contact dermatitis, and postmenopausal osteoporosis. BLT2 plays different roles from BLT1, and one important role of BLT2 is the maintenance of mucosal integrity in the colon.     

VEGF-A stimulation of leukocyte adhesion to colonic microvascular endothelium: implications for inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder characterized by increased leukocyte recruitment and subsequent tissue damage. An increase in the density of the microvasculature of the colon during IBD has been suggested, leading to the concept that angiogenesis may play a pathological role in IBD. Increased tissue and serum levels of the angiogenic cytokine VEGF-A have been reported in cases of active IBD. In this study, we examined the hypothesis that VEGF-A exerts a proinflammatory effect on colon microvascular endothelium that contributes to colonic inflammation. Leukocyte adhesion to VEGF-A-stimulated colon microvascular endothelial cells was examined using a parallel-plate hydrodynamic flow chamber. ICAM-1 adhesion molecule expression on colonic microvascular endothelium also was determined in response to VEGF-A stimulation, along with characterization of leukocyte adhesion molecule expression. High-dose VEGF-A (50 ng/ml) stimulation increased neutrophil and T cell adhesion to and decreased rolling velocities on activated endothelium, whereas low-dose VEGF-A (10 ng/ml) was without effect. Colonic endothelium constitutively expressed ICAM-1, which was significantly increased by treatment with 50 ng/ml VEGF-A or 10 ng/ml TNF-alpha but not 10 ng/ml VEGF-A. T cells expressed CD18 and CD11a with no expression of CD11b, whereas neutrophils expressed CD18, CD11a, and CD11b. Finally, VEGF-A-dependent leukocyte adhesion was found to occur in a CD18-dependent manner. These results demonstrate that VEGF-A levels found in IBD exert a proinflammatory effect similar to other inflammatory agents and suggest that this cytokine may serve as an intermediary between angiogenic stimulation and cell-mediated immune responses.