Transmission of a common intestinal neoplasm in zebrafish by cohabitation.

Intestinal neoplasms are common in zebrafish (Danio rerio) research facilities. These tumours are most often seen in older fish and are classified as small cell carcinomas or adenocarcinomas. Affected fish populations always contain subpopulations with preneoplastic lesions, characterized by epithelial hyperplasia or inflammation. Previous observations indicated that these tumours are unlikely caused by diet, water quality or genetic background, suggesting an infectious aetiology. We performed five transmission experiments by exposure of naïve fish to affected donor fish by cohabitation or exposure to tank effluent water. Intestinal lesions were observed in recipient fish in all exposure groups, including transmissions from previous recipient fish, and moribund fish exhibited a higher prevalence of neoplasms. We found a single 16S rRNA sequence, most similar to Mycoplasma penetrans, to be highly enriched in the donors and exposed recipients compared to unexposed control fish. We further tracked the presence of the Mycoplasma sp. using a targeted PCR test on individual dissected intestines or faeces or tank faeces. Original donor and exposed fish populations were positive for Mycoplasma, while corresponding unexposed control fish were negative. This study indicates an infectious aetiology for these transmissible tumours of zebrafish and suggests a possible candidate agent of a Mycoplasma species.

Androgens regulate prostate cancer cell growth via an AMPK-PGC-1α-mediated metabolic switch.

Prostate cancer is the most commonly diagnosed malignancy among men in industrialized countries, accounting for the second leading cause of cancer-related deaths. Although we now know that the androgen receptor (AR) is important for progression to the deadly advanced stages of the disease, it is poorly understood what AR-regulated processes drive this pathology. Here we demonstrate that AR regulates prostate cancer cell growth via the metabolic sensor 5'-AMP-activated protein kinase (AMPK), a kinase that classically regulates cellular energy homeostasis. In patients, activation of AMPK correlated with prostate cancer progression. Using a combination of radiolabeled assays and emerging metabolomic approaches, we also show that prostate cancer cells respond to androgen treatment by increasing not only rates of glycolysis, as is commonly seen in many cancers, but also glucose and fatty acid oxidation. Importantly, this effect was dependent on androgen-mediated AMPK activity. Our results further indicate that the AMPK-mediated metabolic changes increased intracellular ATP levels and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-mediated mitochondrial biogenesis, affording distinct growth advantages to the prostate cancer cells. Correspondingly, we used outlier analysis to determine that PGC-1α is overexpressed in a subpopulation of clinical cancer samples. This was in contrast to what was observed in immortalized benign human prostate cells and a testosterone-induced rat model of benign prostatic hyperplasia. Taken together, our findings converge to demonstrate that androgens can co-opt the AMPK-PGC-1α signaling cascade, a known homeostatic mechanism, to increase prostate cancer cell growth. The current study points to the potential utility of developing metabolic-targeted therapies directed toward the AMPK-PGC-1α signaling axis for the treatment of prostate cancer.

Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging.

Simultaneous atomic force microscopy (AFM) and confocal fluorescence imaging were used to observe in aqueous buffer the three-dimensional landscape of the inner surface of membrane sheets stripped from fixed tumor mast cells. The AFM images reveal prominent, irregularly shaped raised domains that label with fluorescent markers for both resting and activated immunoglobin E receptors (FcepsilonRI), as well as with cholera toxin-aggregated GM1 and clathrin. The latter suggests that coated pits bud from these regions. These features are interspersed with flatter regions of membrane and are frequently surrounded and interconnected by cytoskeletal assemblies. The raised domains shrink in height by approximately 50% when cholesterol is extracted with methyl-beta-cyclodextrin. Based on composition, the raised domains seen by AFM correspond to the cholesterol-enriched dark patches observed in transmission electron microscopy (TEM). These patches were previously identified as sites of signaling and endocytosis based on their localization of activated FcepsilonRI, at least 10 associated signaling molecules, and the presence of clathrin-coated pits. Overall the data suggest that signaling and endocytosis occur in mast cells from raised membrane regions that depend on cholesterol for their integrity and may be organized in specific relationship with the cortical cytoskeleton.

Local mobility in lipid domains of supported bilayers characterized by atomic force microscopy and fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is used to examine mobility of labeled probes at specific sites in supported bilayers consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid domains in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Those sites are mapped beforehand with simultaneous atomic force microscopy and submicron confocal fluorescence imaging, allowing characterization of probe partitioning between gel DPPC and disordered liquid DOPC domains with corresponding topography of domain structure. We thus examine the relative partitioning and mobility in gel and disordered liquid phases for headgroup- and tailgroup-labeled GM1 ganglioside probes and for headgroup- and tailgroup-labeled phospholipid probes. For the GM1 probes, large differences in mobility between fluid and gel domains are observed; whereas unexpected mobility is observed in submicron gel domains for the phospholipid probes. We attribute the latter to domain heterogeneities that could be induced by the probe. Furthermore, fits to the FCS data for the phospholipid probes in the DOPC fluid phase require two components (fast and slow). Although proximity to the glass substrate may be a factor, local distortion of the probe by the fluorophore could also be important. Overall, we observe nonideal aspects of phospholipid probe mobility and partitioning that may not be restricted to supported bilayers.

Exclusive neutrophil recruitment with oncostatin M in a human system.

Oncostatin M (OSM), a member of the IL-6 family has been postulated to be a potent recruiter of leukocytes, however information regarding the molecular mechanism(s) underlying this event is extremely limited. Therefore, the aim of this study was to investigate the role of OSM-mediated leukocyte recruitment in a human system in vitro under flow conditions. A parallel-plate flow chamber assay was used to examine leukocyte recruitment from whole blood by human umbilical vein endothelium treated for 24 hours with OSM. OSM in a dose-response manner revealed very significant leukocyte rolling and adhesion reaching optimal levels at a very low concentration of OSM (10 ng/ml). The OSM-induced leukocyte rolling and adhesion was comparable to levels seen with tumor necrosis factor. OSM was extremely selective for neutrophil recruitment (96%) with <3% lymphocyte recruitment. By contrast, tumor necrosis factor-alpha revealed no such selectivity, recruiting 70% neutrophils and at least 25% lymphocytes and detectable levels of eosinophils at 24 hours. The molecular mechanism underlying the leukocyte recruitment seemed to be entirely dependent on P-selectin as leukocyte recruitment could be completely blocked by the addition of a P-selectin-blocking antibody. An elevation in both P-selectin message and protein was observed with 24 hours of OSM stimulation of endothelium. By contrast, E-selectin and VCAM-1 were not detectable after OSM stimulation. Similar results were seen with passaged dermal microvascular endothelium that does not have a prestored pool of P-selectin. Based on these results, we conclude that OSM may be a very selective potent recruiter of neutrophils in more prolonged inflammatory conditions, an event exclusively dependent on P-selectin.

The role of Mac-1 (CD11b/CD18) in antigen-induced airway eosinophilia in mice.

Mac-1 (CD11b/CD18) is an important adhesion molecule involved in the migration of leukocytes, cell signaling, and subsequent secretory responses. Its precise role in eosinophil recruitment and activation in vivo is not entirely clear. We wished to directly examine the role of Mac-1 in eosinophil migration in a murine model of allergic pulmonary inflammation. Briefly, wild-type (C57Bl/6) and Mac-1-deficient/knockout (Mac-1 KO) mice were intraperitoneally sensitized with ovalbumin (OVA) and alum (AlOH) on Days 0 and 14, and intranasally challenged with OVA either once on Day 14 or five times on Days 14 and 25 through 28. Control animals were challenged with saline. Bronchial hyperresponsiveness was measured, bronchoalveolar lavage (BAL) fluid was collected, and lungs were harvested for histology 24 h after the last challenge. The data demonstrate that wild-type (WT) mice do not respond to one OVA challenge but do develop bronchial hyperreactivity and airway and tissue eosinophilia after five OVA challenges. Conversely, Mac-1 KO mice develop significant airway eosinophilia after one OVA challenge, and the degree of airway inflammation is comparable to that observed in allergic WT mice after five challenges. In Mac-1 KO mice, after five challenges, bronchial hyperreactivity and airway inflammation was significantly enhanced compared with their wild-type counterparts. Administration of an anti-Mac-1 antibody to WT mice, before each of five intranasal OVA challenges, significantly reduces the airway eosinophilia but has no effect on tissue eosinophilia or bronchial hyperresponsiveness. Intravenous injection of interleukin-5 induced a significant blood eosinophilia in both WT and Mac-1 KO mice. Intranasal eotaxin administration induced similar levels of eosinophil migration into the lung tissues and airways of both WT and Mac-1 KO mice. In conclusion, Mac-1-deficient mice develop enhanced eosinophilic inflammation in the lung in response to allergic antigen challenge.

HIV-1 penetrates coronary artery endothelial cells by transcytosis.

BACKGROUND: The pathogenesis of HIV-1-related cardiomyopathy is poorly understood, but HIV-1 has been detected in cardiomyocytes. Whether HIV-1 penetrates into the myocardium by infection of coronary artery endothelial cells (CAEC) or using transcellular or paracellular routes across CAEC has not been resolved. MATERIALS AND METHODS: A model of the CAEC barrier was constructed with primary CAEC (derived from human coronary vessels). Polymerase chain reaction (PCR) assay, infectious assay, and immunofluorescence were employed to show abortive nature of HIV-1 infection of CAEC. Tight junction (TJ) and cell adhesion proteins were visualized by immunofluorescence. The time course of HIV-1 invasion was measured by HIV-1 RNA assay. Inulin permeability assay determined paracellular leakage. Transmission electron microscopy demonstrated virus-induced endothelial vacuolization. RESULTS: Despite a strong display on CAEC of CXCR4 and a lesser expression of CCR3 and CCR5, HIV-1 did not productively replicate in CAEC, as shown by infectious assay, immunofluorescence, and electron microscopy. HIV-1 infection of CAEC was abortive with minimal reverse transcription of strong stop DNA and pol but not full-length or two LTR DNA circles. Upon infection of the model with 1 million RNA copies of HIV-1JR-FL, virus penetration 2 hr postinfection (PI) was negligible but increased by 1,750% 24 hr PI. The paracellular permeability increased during this period by only 25%. Neither AOP-RANTES nor v-MIPII significantly reduced HIV-1JR-FL invasion. Virus infection did not alter the integral TJ protein occludin and the TJ-associated protein ZO-1. HIV-1 exposed CAEC and brain microvascular endothelial cells (BMVEC) developed extensive cytoplasmic vacuolization with retroviral-like particles in the vacuoles. CONCLUSIONS: The endothelium is not an impenetrable barrier to HIV-1. The virus opens a transcellular route across coronary and brain endothelia in cytoplasmic vacuoles.

Matrix-dependent mechanism of neutrophil-mediated release and activation of matrix metalloproteinase 9 in myocardial ischemia/reperfusion.

BACKGROUND: A key component of reperfusion of myocardial infarction is an immediate inflammatory response, which enhances tissue repair. Matrix turnover is crucial to tissue repair, and matrix metalloproteinases (MMPs) are key enzymes involved in matrix degradation. The hypothesis tested is that one inflammation-based effector of tissue repair is the secretion and activation of MMP-9 by infiltrating neutrophils. METHODS AND RESULTS: Cardiac lymph and tissue were assayed for atent and active MMP-2 and MMP-9 by zymography and immunochemistry. Dual-labeling immunofluorescence determined the cellular source of MMP-9 protein. Isolated canine neutrophils were incubated with preischemic and postischemic cardiac lymph in the presence and absence of collagen-fibronectin pads, and the supernatants were assayed for latent and active MMP-9. MMP-9 increased during the first hours of reperfusion in both lymph supernatants and myocardial extracts, and this increase was of neutrophil origin. MMP-9 in the cardiac lymph remained latent but was activatable. In contrast, MMP-9 in the myocardium was in both latent and active forms. In situ zymography demonstrated that activated MMP-9 surrounded the infiltrated neutrophils. When postischemic cardiac lymph was incubated with neutrophils in vitro, MMP-9 secretion and activation occurred only in the presence of a collagen-fibronectin substrate; preischemic cardiac lymph did not induce significant secretion or activation. CONCLUSIONS: Infiltrating neutrophils are an early source of MMP-9 after reperfusion, and a portion of MMP-9 in the myocardium is active. Infiltrating neutrophils may localize MMP-9 activation by secreting MMP-9 and as a source of activating proteases.

Self-assembly of mesoscopically ordered chromatic polydiacetylene/silica nanocomposites.

Nature abounds with intricate composite architectures composed of hard and soft materials synergistically intertwined to provide both useful functionality and mechanical integrity. Recent synthetic efforts to mimic such natural designs have focused on nanocomposites, prepared mainly by slow procedures like monomer or polymer infiltration of inorganic nanostructures or sequential deposition. Here we report the self-assembly of conjugated polymer/silica nanocomposite films with hexagonal, cubic or lamellar mesoscopic order using polymerizable amphiphilic diacetylene molecules as both structure-directing agents and monomers. The self-assembly procedure is rapid and incorporates the organic monomers uniformly within a highly ordered, inorganic environment. Polymerization results in polydiacetylene/silica nanocomposites that are optically transparent and mechanically robust. Compared to ordered diacetylene-containing films prepared as Langmuir monolayers or by Langmuir-Blodgett deposition, the nanostructured inorganic host alters the diacetylene polymerization behaviour, and the resulting nanocomposite exhibits unusual chromatic changes in response to thermal, mechanical and chemical stimuli. The inorganic framework serves to protect, stabilize, and orient the polymer, and to mediate its function. The nanocomposite architecture also provides sufficient mechanical integrity to enable integration into devices and microsystems.

alpha(4)-integrin mediates neutrophil-induced free radical injury to cardiac myocytes.

Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via beta(2)-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the alpha(4)-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) beta(2)-integrins (CD18) still signal oxidant production, or if this process is now coupled to the alpha(4)-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte-PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30-50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both alpha(4)-integrin antibody (Ab)-treated PMNs and NADPH oxidase-deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti-alpha(4)-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the alpha(4)-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed alpha(4)-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the alpha(4)-integrin-coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.

Pathophysiologic importance of E- and L-selectin for neutrophil-induced liver injury during endotoxemia in mice.

Neutrophils can cause parenchymal cell injury in the liver during ischemia-reperfusion and endotoxemia. Neutrophils relevant for the injury accumulate in sinusoids, transmigrate, and adhere to hepatocytes. To investigate the role of E- and L-selectin in this process, C3Heb/FeJ mice were treated with 700 mg/kg galactosamine and 100 microgram/kg endotoxin (Gal/ET). Immunogold labeling verified the expression of E-selectin on sinusoidal endothelial cells 4 hours after Gal/ET injection. In addition, Gal/ET caused up-regulation of Mac-1 (CD11b/CD18) and shedding of L-selectin from circulating neutrophils. Gal/ET induced hepatic neutrophil accumulation (422 +/- 32 polymorphonuclear leukocytes [PMN]/50 high power fields [HPF]) and severe liver injury (plasma alanine transaminase [ALT] activities: 4,120 +/- 960 U/L; necrosis: 44 +/- 3%) at 7 hours. Treatment with an anti-E-selectin antibody (3 mg/kg, intravenously) at the time of Gal/ET administration did not significantly affect hepatic neutrophil accumulation and localization. However, the anti-E-selectin antibody significantly attenuated liver injury as indicated by reduced ALT levels (-84%) and 43% less necrotic hepatocytes. In contrast, animals treated with an anti-L-selectin antibody or L-selectin gene knock out mice were not protected against Gal/ET-induced liver injury. However, E-, L-, and P-selectin triple knock out mice showed significantly reduced liver injury after Gal/ET treatment as indicated by lower ALT levels (-65%) and reduced necrosis (-68%). Previous studies showed that circulating neutrophils of E-selectin-overexpressing mice are primed and activated similar to neutrophils adhering to E-selectin in vitro. Therefore, we conclude that blocking E-selectin or eliminating this gene may have protected against Gal/ET-induced liver injury in vivo by inhibiting the full activation of neutrophils during the transmigration process.

Preferential sites for stationary adhesion of neutrophils to cytokine-stimulated HUVEC under flow conditions.

Neutrophils form CD18-dependent adhesions to endothelial cells at sites of inflammation. This phenomenon was investigated under conditions of flow in vitro using isolated human neutrophils and monolayers of HUVEC. The efficiency of conversion of neutrophil rolling to stable adhesion in this model was >95%. Neither anti-CD11a nor anti-CD11b antibodies significantly altered the extent of this conversion, but a combination of both antibodies inhibited the arrest of rolling neutrophils by >95%. The efficiency of transendothelial migration of arrested neutrophils was >90%, and the site of transmigration was typically <6 microm from the site of stationary adhesion. Approximately 70% of transmigrating neutrophils migrated at tricellular corners between three adjacent endothelial cells. A model of neutrophils randomly distributed on endothelium predicted a significantly greater migration distance to these preferred sites of transmigration, but a model of neutrophils adhering to endothelial borders is consistent with observed distances. It appears that stable adhesions form very near tricellular corners.

Increased sensitivity to the C-X-C chemokine CINC/gro in a model of chronic inflammation.

OBJECTIVE: The C-C chemokine MCP-1 elicits significant neutrophil emigration in rats with chronic adjuvant-induced inflammation, but not in naive animals. We examined responses to the C-X-C chemokine CINC/gro to determine whether this class of chemokine elicits altered neutrophil responses during chronic inflammation. METHODS: CINC/gro was superfused over mesenteric venules of naive rats or animals with chronic adjuvant-induced vasculitis. Antibodies were used to characterize adhesive mechanisms. RESULTS: CINC/gro elicited leukocyte transendothelial migration in adjuvant-immunized rats at 100-fold lower concentrations than required to elicit transmigration in naive animals. In both groups, neutrophils constituted > 95% of the leukocytes recruited by CINC/gro. Using in vitro chemotaxis assays, neutrophils from control and adjuvant-immunized rats responded equally to CINC/gro, suggesting differences in migration were not related to neutrophil phenotype. Differences in adhesion molecule usage were noted in vivo. In control animals, CD18 antibodies blocked CINC/gro-induced neutrophil adhesion and emigration. In adjuvant-immunized animals, an alpha 4-integrin antibody reduced adhesion and emigration, while a CD18 antibody selectively inhibited emigration. CONCLUSIONS: This study demonstrates increased sensitivity to a C-X-C chemokine in a model of chronic inflammation, implicates the alpha 4-integrin in neutrophil adhesion, and demonstrates that CD18 mediates leukocyte transendothelial migration independent from firm adhesion.

Analysis of tight junctions during neutrophil transendothelial migration.

Intercellular junctions have long been considered the main sites through which adherent neutrophils (PMNs) penetrate the endothelium. Tight junctions (TJs; zonula occludens) are the most apical component of the intercellular cleft and they form circumferential belt-like regions of intimate contact between adjacent endothelial cells. Whether PMN transmigration involves disruption of the TJ complex is unknown. We report here that endothelial TJs appear to remain intact during PMN adhesion and transmigration. Human umbilical vein endothelial cell (HUVEC) monolayers, a commonly used model for studying leukocyte trafficking, were cultured in astrocyte-conditioned medium to enhance TJ expression. Immunofluorescence microscopy and immunoblot analysis showed that activated PMN adhesion to resting monolayers or PMN migration across interleukin-1-treated monolayers does not result in widespread proteolytic loss of TJ proteins (ZO-1, ZO-2, and occludin) from endothelial borders. Ultrastructurally, TJs appear intact during and immediately following PMN transendothelial migration. Similarly, transendothelial electrical resistance is unaffected by PMN adhesion and migration. Previously, we showed that TJs are inherently discontinuous at tricellular corners where the borders of three endothelial cells meet and PMNs migrate preferentially at tricellular corners. Collectively, these results suggest that PMN migration at tricellular corners preserves the barrier properties of the endothelium and does not involve widespread disruption of endothelial TJs.

Two components of actin-based retrograde flow in sea urchin coelomocytes.

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase-specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a "push-pull" mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

Histochemical and morphological characteristics of canine cardiac mast cells.

Cardiac mast cells have been recently isolated and characterized in humans, however canine cardiac mast cells have not been investigated. The objective of this study is to describe the histological and morphological characteristics of canine cardiac mast cells and examine the potential usefulness of canine models in investigating the role of mast cells in cardiovascular pathology. Canine cardiac mast cells could be easily identified by staining with Toluidine Blue or FITC-avidin. Using Toluidine Blue staining, we demonstrated fewer mast cells in formalin-fixed samples than in specimens fixed in Carnoy's, thus identifying a formalin-sensitive mast cell population in the canine heart. Mast cells were equally distributed in atria and ventricles with approximately 50% showing a perivascular location. Using enzyme-histochemical techniques, we detected tryptase and chymase activity in canine cardiac mast cells. Ultrastructural studies identified mast cells as granular cells with an eccentric non-segmented nucleus. Immunohistochemistry with the macrophage specific antibody AM-3K demonstrated that resident cardiac macrophages were 1.9 times more numerous than mast cells, also showing a predominantly perivascular (60%) location. Perivascular macrophages were more often periarteriolar, whereas perivascular mast cells were more often located along small veins and capillaries. Due to their ability to release cytokines and growth factors and their strategic perivascular location, resident cardiac inflammatory cells, such as mast cells and macrophages, may be important in pathological processes causing myocardial inflammation and fibrosis. Furthermore, mast cell-derived chymase, an important angiotensin II-forming enzyme may have a significant role in regulating the cardiac renin-angiotensin system.

Chronic inflammation upregulates chemokine receptors and induces neutrophil migration to monocyte chemoattractant protein-1.

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that stimulates monocyte recruitment when injected into tissues of healthy animals. However, the function of this chemokine in models with preexisting inflammation is not known. Therefore, MCP-1 was superfused over the mesentery of naive rats or rats with chronic adjuvant-induced vasculitis. MCP-1 elicited increased leukocyte transendothelial migration in adjuvant-immunized rats compared with naive animals. Surprisingly, histology revealed that neutrophils constituted the majority of leukocytes recruited in adjuvant-immunized animals. In vitro, MCP-1 was also able to induce chemotaxis of neutrophils isolated from adjuvant-immunized rats but not from naive rats. Flow cytometry revealed novel expression of the CC chemokine receptors CCR1 and CCR2 on neutrophils from adjuvant-immunized animals. In naive animals, an antibody against CD18 blocked leukocyte adhesion and emigration in response to MCP-1. In adjuvant-immunized animals, leukocyte adhesion was reduced by antibodies against the alpha4-integrin but not by antibodies against CD18. However, the CD18 antibody did block emigration. To our knowledge, this study is the first to show increased sensitivity to a CC chemokine in a model with preexisting inflammation, and altered leukocyte recruitment profiles in response to MCP-1. It also demonstrates that CD18 is required for chemokine-induced leukocyte transendothelial migration, independent of its known role in mediating firm adhesion. J. Clin. Invest. 103:1269-1276 (1999).

P-selectin mediates neutrophil adhesion to endothelial cell borders.

During an acute inflammatory response, endothelial P-selectin (CD62P) can mediate the initial capture of neutrophils from the free flowing bloodstream. P-selectin is stored in secretory granules (Weibel-Palade bodies) and is rapidly expressed on the endothelial surface after stimulation with histamine or thrombin. Because neutrophil transmigration occurs preferentially at endothelial borders, we wished to determine whether P-selectin-dependent neutrophil capture (adhesion) occurs at endothelial cell borders. Under static or hydrodynamic flow (2 dyn/cm2) conditions, histamine (10(-4) M) or thrombin (0.2 U/mL) treatment induced preferential (> or = 75%) neutrophil adhesion to the cell borders of endothelial monolayers. Blocking antibody studies established that neutrophil adhesion was completely P-selectin dependent. P-selectin surface expression increased significantly after histamine treatment and P-selectin immunostaining was concentrated along endothelial borders. We conclude that preferential P-selectin expression along endothelial borders may be an important mechanism for targeting neutrophil migration at endothelial borders.

CXC and CC chemokine receptors on coronary and brain endothelia.

BACKGROUND: Chemokine receptors on leukocytes play a key role in inflammation and HIV-1 infection. Chemokine receptors on endothelia may serve an important role in HIV-1 tissue invasion and angiogenesis. MATERIALS AND METHODS: The expression of chemokine receptors in human brain microvascular endothelial cells (BMVEC) and coronary artery endothelial cells (CAEC) in vitro and cryostat sections of the heart tissue was determined by light and confocal microscopy and flow cytometry with monoclonal antibodies. Chemotaxis of endothelia by CC chemokines was evaluated in a transmigration assay. RESULTS: In BMVEC, the chemokine receptors CCR3 and CXCR4 showed the strongest expression. CXCR4 was localized by confocal microscopy to both the cytoplasm and the plasma membrane of BMVEC. In CAEC, CXCR4 demonstrated a strong expression with predominantly periplasmic localization. CCR5 expression was detected both in BMVEC and CAEC but at a lower level. Human umbilical cord endothelial cells (HUVEC) expressed strongly CXCR4 but only weakly CCR3 and CCR5. Two additional CC chemokines, CCR2A and CCR4, were detected in BMVEC and CAEC by immunostaining. Immunocytochemistry of the heart tissues with monoclonal antibodies revealed a high expression of CXCR4 and CCR2A and a low expression of CCR3 and CCR5 on coronary vessel endothelia. Coronary endothelia showed in vitro a strong chemotactic response to the CC chemokines RANTES, MIP-1alpha, and MIP-1beta. CONCLUSIONS: The endothelia isolated from the brain display strongly both the CCR3 and CXCR4 HIV-1 coreceptors, whereas the coronary endothelia express strongly only the CXCR4 coreceptor. CCR5 is expressed at a lower level in both endothelia. The differential display of CCR3 on the brain and coronary endothelia could be significant with respect to the differential susceptibility of the heart and the brain to HIV-1 invasion. In addition, CCR2A is strongly expressed in the heart endothelium. All of the above chemokine receptors could play a role in endothelial migration and repair.

Endothelial cell "memory" of inflammatory stimulation: human venular endothelial cells store interleukin 8 in Weibel-Palade bodies.

The expression and secretion of interleukin (IL)-8, the prototype member of the C-X-C subfamily of chemokines, can be induced by diverse inflammatory stimuli in many cells, including endothelial cells (EC). Upon de novo synthesis, IL-8 localizes intracellularly in the Golgi apparatus, from where it is secreted. In addition to this constitutive secretory pathway, we describe a depot storage and separate regulated secretory pathway of IL-8 in EC. The prolonged stimulation of primary human EC with inflammatory mediators resulted in the accumulation of IL-8 in Weibel-Palade bodies, where it colocalized with von Willebrand factor. IL-8 was retained in these storage organelles for several days after the removal of the stimulus and could be released by EC secretagogues such as phorbol myristate acetate, the calcium ionophore A23187, and histamine. These findings suggest that storage of IL-8 in Weibel-Palade bodies may serve as the EC "memory" of a preceding inflammatory insult, which then enables the cells to secrete IL-8 immediately without de novo protein synthesis.