Role of CD44 and hyaluronan in neutrophil recruitment

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44(-/-) and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44(-/-) mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44(-/-) mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44(-/-) mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44(-/-) mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.     

Generation of, lipid neutrophil chemoattractant activity by histamine-stimulated cultured endothelial cells

Endothelial cell-neutrophil interactions are an important aspect of inflammatory responses. Because vascular endothelial cells respond to the inflammatory mediator histamine, these studies determined whether histamine could induce endothelial cells to release substances that affect human neutrophil migration. Cultured bovine and human endothelial cells incubated with histamine released neutrophil chemoattractant activity within 1 min; peak levels were noted in 45 min. Cimetidine, an H2 receptor antagonist, blocked chemoattractant production, whereas diphenhydramine, an H1 receptor antagonist, did not. Cycloheximide did not inhibit release of chemoattractant activity, suggesting de novo protein synthesis was not necessary for its appearance. Extraction with acidified diethyl ether partitioned all neutrophil chemoattractant activity into the organic phase. The lipoxygenase pathway inhibitors, diethylcarbamazine and 5,8,11,14 eicosatetraynoic acid, inhibited generation of this lipophilic chemoattractant activity, whereas indomethacin, a cyclo-oxygenase inhibitor, did not. Resolution of the histamine-induced endothelial cell-derived chemoattractant activity by reverse-phase high pressure liquid chromatography yielded several peaks of chemoattractant activity, none of which co-eluted with leukotriene B4, platelet-activating factor, or two mono-hydroxyeicostetraenoic acids. These findings suggest that endothelial cells release lipid neutrophil chemoattractant activity that may play a role in inflammatory responses associated with histamine.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis

Leukocyte infiltration into the liver is paramount to the development of liver injury in hepatitis. Hepatitis occurring after the administration of Con A in mice is felt to be a T lymphocyte-mediated disease. In this study, we report that neutrophils are the key initiators of lymphocyte recruitment and liver injury caused by Con A. The objectives of this study were to investigate the involvement of neutrophils in Con A-induced hepatitis in vivo via intravital microscopy. After Con A administration, we observed a significant increase in leukocyte rolling flux, a decrease in rolling velocity, and an increase in leukocyte adhesion to the hepatic microvasculature. Fluorescence microscopy identified that within 4 h of Con A administration only a minority of the recruited leukocytes were T lymphocytes. Furthermore, immunohistochemistry showed a significant increase in neutrophils recruited to the liver post-Con A treatment in association with liver cell damage, as reflected by elevated serum alanine aminotransferase levels. Using flow cytometry, we observed that Con A could bind directly to neutrophils, which resulted in a shedding of L-selectin, an increase in beta(2)-integrin expression, and the production of reactive oxidants. Following neutrophil depletion, a significant inhibition of Con A-induced CD4+ T lymphocyte recruitment to the liver resulted and complete reduction in hepatic injury, as assessed by serum alanine aminotransferase levels. In summary, the present data support the concept that neutrophils play an important and previously unrecognized role in governing Con A-induced CD4+ T cell recruitment to the liver and the subsequent development of hepatitis.     

Tilapia mast cell lysates enhance neutrophil adhesion to cultured vascular endothelial cells

The possible role of fish mast cells in regulating neutrophil adhesion to vascular endothelial cells was studied using primary cultures of tilapia vascular endothelial cells. The endothelial cell monolayer, which was cultured in 96 well plates, was stimulated for appropriate periods with tilapia mast cell (tMC)-lysates or with Leibovitz-15 (L-15) medium, as a control, and peripheral neutrophils were added into each well after removal of the lysates. After 30 min incubation, cells in the wells were fixed with formalin and non-adherent neutrophils were removed. The cells were stained with Giemsa and neutrophil adhesion was observed microscopically. Although some neutrophils attached to the endothelial cells without stimulation, neutrophil adhesion was enhanced after the incubation of the endothelial cells with tMC-lysates. Neutrophil adhesion was maximal 6 h after the lysate stimulation, with a six-fold increase compared to the control. Neutrophil adhesion also increased when the endothelial cells were stimulated with neutrophil lysates, lipopolysaccharide and zymosan-treated tilapia sera. These results indicate that fish vascular endothelial cells express some neutrophil adhesion molecule(s) after stimulation with various substances.     

Tissue- and stimulus-dependent role of phosphatidylinositol 3-kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo

PI3K plays a fundamental role in regulating neutrophil recruitment into sites of inflammation but the role of the different isoforms of PI3K remains unclear. In this study, we evaluated the role of PI3Kgamma and PI3Kdelta for neutrophil influx induced by the exogenous administration or the endogenous generation of the chemokine CXCL1. Administration of CXCL1 in PI3Kgamma(-/-) or wild-type (WT) mice induced similar increases in leukocyte rolling, adhesion, and emigration in the cremaster muscle when examined by intravital microscopy. The induction of neutrophil recruitment into the pleural cavity or the tibia-femoral joint induced by the injection of CXCL1 was not significantly different in PI3Kgamma(-/-) or WT mice. Neutrophil influx was not altered by treatment of WT mice with a specific PI3Kdelta inhibitor, IC87114, or a specific PI3Kgamma inhibitor, AS605240. The administration of IC87114 prevented CXCL1-induced neutrophil recruitment only in presence of the PI3Kgamma inhibitor or in PI3Kgamma(-/-) mice. Ag challenge of immunized mice induced CXCR2-dependent neutrophil recruitment that was inhibited by wortmannin or by blockade of and PI3Kdelta in PI3Kgamma(-/-) mice. Neutrophil recruitment to bronchoalveolar lavage induced by exogenously added or endogenous production of CXCL1 was prevented in PI3Kgamma(-/-) mice. The accumulation of the neutrophils in lung tissues was significantly inhibited only in PI3Kgamma(-/-) mice treated with IC87114. Neutrophil recruitment induced by exogenous administration of C5a or fMLP appeared to rely solely on PI3Kgamma. Altogether, our data demonstrate that there is a tissue- and stimulus-dependent role of PI3Kgamma and PI3Kdelta for neutrophil recruitment induced by different chemoattractants in vivo.     

Basal lamina formation by cultured microvascular endothelial cells

The production of a basal lamina by microvascular endothelial cells (MEC) cultured on various substrata was examined. MEC were isolated from human dermis and plated on plastic dishes coated with fibronectin, or cell-free extracellular matrices elaborated by fibroblasts, smooth muscle cells, corneal endothelial cells, or PF HR9 endodermal cells. Examination of cultures by electron microscopy at selected intervals after plating revealed that on most substrates the MEC produced an extracellular matrix at the basal surface that was discontinuous, multilayered, and polymorphous. Immunocytochemical studies demonstrated that the MEC synthesize and deposit both type IV collagen and laminin into the subendothelial matrix. When cultured on matrices produced by the PF HR9 endodermal cells MEC deposit a subendothelial matrix that was present as a uniform sheet which usually exhibited lamina rara- and lamina densa-like regions. The results indicate that under the appropriate conditions, human MEC elaborate a basal lamina-like matrix that is ultrastructurally similar to basal lamina formed in vivo, which suggests that this experimental system may be a useful model for studies of basal lamina formation and metabolism.     

Neovascular responses induced by cultured aortic endothelial cells

Neovascularization was studied in the chorioallantoic membrane of the chick embryo after implantation of bovine aortic endothelial and smooth muscle cells, Swiss and BALB/c 3T3 cells and human diploid fibroblasts cultured separately on microcarrier beads. Quantitative analysis of neovascularization indicated a 3 1/2-fold increase in the number of blood vessels responding to endothelial cells while smooth muscle cells induced a twofold increase when compared to the response of beads without cells. Skin fibroblasts and Swiss 3T3 cells did not elicit a comparable response. The marked angiogenic response induced by endothelial cells was characterized by a 137% increase in total vessel length and a 35% increase in average vessel area when compared to controls. Two of the properties required for an angiogenesis factor--stimulation of cellular migration and proliferation--can also be demonstrated using endothelial cell-conditioned medium in cell culture systems. Medium from cultured bovine aortic endothelium stimulates DNA synthesis, proliferation, and migration of smooth muscle cells. In addition, conditioned media from both endothelial cells and smooth muscle cells produced an angiogenic response in the chorioallantoic membrane assay, which was comparable to that produced by intact cells growing on microcarrier beads. Similar responses were not evident with medium conditioned by other cell types. These results indicate the potential importance of endothelial cells and endothelial cell products in regulating blood vessel growth.     

Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells

Bovine corneal endothelial (BCE) cells seeded and grown on plastic dishes were labeled with 35S-sulfate or 3H-glucosamine for 48 h at various phases of growth of the cultures. Newly synthesized proteoglycans were isolated from the culture medium and from the extracellular matrix (ECM) produced by the BCE cells, and the glycosaminoglycan (GAG) component of the proteoglycans was analyzed. Cells actively proliferating on plastic surfaces secreted an ECM that contained heparan sulfate as the major 35S-labeled GAG (86%) and dermatan sulfate as a minor component (13%). Upon reaching confluence, the BCE cells incorporated 35S-labeled chondroitin sulfate (20%), as well as heparan sulfate (66%) and dermatan sulfate (14%), into the EC. Seven-day postconfluent cells incorporated newly synthesized heparan sulfate and dermatan sulfate into the matrix in approximately equal proportions. Dermatan sulfate was the main 35S-labeled GAG (60-65%) in the medium of both confluent and postconfluent cultures. 35S-Labeled chondroitin sulfate (20-25%) and heparan sulfate (15%) were also secreted into the culture medium. The type of GAG incorporated into newly synthesized ECM was affected when BCE cells were seeded onto ECM-coated dishes instead of plastic. BCE cells actively proliferating on ECM-coated dishes incorporated newly synthesized heparan sulfate and dermatan sulfate into the ECM in a ratio that was very similar to the ratio of these GAGs in the underlying ECM. Addition of mitogens such as fibroblast growth factor (FGF) to the culture medium altered the type of GAG synthesized and incorporated into the ECM by BCE cells seeded onto ECM-coated dishes if the cells were actively growing, but had no effect on postconfluent cultures.     

Protein phosphorylation in cultured endothelial cells

We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP-dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP-dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin-dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.     

Characterization of phospholipase A2 activation by plasmin in cultured bovine endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 0.1 µM human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A₂ with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A₂ by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A₂ from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A₂ in endothelial cells.     

Morphological alterations in cultured endothelial cells induced by arachidonic acid

The addition of arachidonic acid (20:4), but not other fatty acids, including the structurally similar eicosapentaenoic acid (20:5), induced specific morphological changes in cultured endothelial cells derived from bovine aorta and pulmonary artery. Cells exhibited a time- and dose-dependent change from their normal, epithelioid morphology to become elongated, polygonal, and spindle-shaped. Cells isolated from aorta appeared more sensitive to these changes than those from pulmonary artery. The effect was observed as early as 12 h after exposure to 20:4, required 48 h for maximal expression, and could be reversed in 2-5 h after change to normal media. The morphological alteration was not observed in cells treated with leukotrienes or PGE2. When cells were pretreated with ibuprofen, aspirin, or indomethacin to block prostaglandin synthesis and then exposed to 20:4, the dose-response effect was shifted to the left. This increased sensitivity to 20:4 suggests either a direct effect of 20:4 on cell morphology or an indirect effect due to metabolites of 20:4 which are not dependent on the cyclooxygenase pathway.     

State-dependent calcium mobilization by urotensin-II in cultured human endothelial cells

Human endothelial cells express urotensin-II (U-II) as well as its receptor GPR14. Using microfluorimetric techniques, the effect of human U-II on cytosolic Ca2+ concentrations [Ca2+]i in cultured human aortic endothelial cells (HAECs) loaded with Fura-2 was evaluated in static or flow conditions. Under the static state, U-II (100 nM) abolished spontaneous Ca2+ oscillations, which occurred in a population of cultured HAEC. Similarly, U-II reduced thrombin-, but not ATP-induced calcium responses, suggesting that the peptide does not alter the Gq/11/IP3 pathway; rather, it modifies the coupling between protease-activated receptors and Gq/11/IP3. Under the flow condition, U-II (1, 10 and 100 nM) produced a dose-dependent increase in [Ca2+]i, which was subjected to desensitization. The result demonstrates a state-dependent effect of U-II in cultured HAEC, which may explain the variable responses to U-II under different experimental conditions.     

Prostacyclin release induced by neurokinins in cultured human endothelial cells

The effects of neurokinins (NK) and related peptides on the secretion of 6-keto-prostaglandin F1 alpha, a stable metabolite of prostacyclin, were measured. These peptides enhanced three- to five-fold the basal secretion rate with the following rank order of potency (based on threshold concentrations for a significant output): substance P (SP) greater than or equal to NKA greater than SP 4-11 greater than or equal to [pGlu6]SP 6-11 = SP 7-11.NKB and SP 1-9 were inactive. Ac[Arg6, Sar9, Met(O2)11]SP, a NK1 receptor selective agonist, was more potent than other selective agonists for the NK2 and NK3 receptor subtypes. These results suggest that the NK receptors, which mediate the release of prostacyclin from human endothelial cells, belong to the NK1 subtype.     

The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells

The influence of the two surface structures of Neisseria meningitidis, capsule and pili, in bacterial interactions with human endothelial cells was investigated. Increased association correlated with the presence of pili on bacteria while capsule type had no apparent effect. Strains expressing both Class I and Class II pili associated with endothelial cells in significantly larger numbers compared with the non-piliated variants of the same strains (greater than 10x). Variants of Neisseria gonorrhoeae strain P9 expressing antigenically distinct pili also associated with endothelial cells in larger numbers (greater than 30x) compared with the non-piliated variant. Electron microscopic studies confirmed these data and showed that gonococci were internalized more frequently compared with meningococci. One consequence of increased association was an increase in the cytopathic effect of bacteria on the target cells.     

A flow system for the study of shear forces upon cultured endothelial cells

A parallel plate chamber in a flow system has been designed to study the effects of fluid shear stresses on cells. The system was applied to the study of cultured endothelial cells grown on cover slips which were accommodated in recessed wells in the base plate. Dye injection studies in the chamber indicated laminar flow over the cells. Shear rates measured over the cover slips by an electrochemical technique were found to be linear with flow rate. Laser doppler anemometry showed parabolic profiles between the plates. Endothelial cells subjected to flow showed a correlation between the time required for orientation and the magnitude of the shear stress.     

Relation between hyaluronan and sulphated glycosaminoglycan synthesis and degradation in cultured embryonic fibroblasts. Effect of concanavalin A and ammonium chloride administration.

In order to evaluate the relationship between glycosaminoglycan (GAG) synthesis and degradation, the effect of NH4Cl, which inhibits lysosomal degradation, on GAG production was analysed in vitro in concanavalin A (Con A) stimulated fibroblasts from 7 and 14-day-old chick embryos. 35SO4 incorporation into total proteoglycan (PG), 3H incorporation into individual GAG classes, beta-N-acetyl-D-glucosaminidase and beta-D-glucuronidase activity were determined. The results indicate a correlation between Con A and NH4Cl effects: NH4Cl induced a reduction principally in the GAG classes most stimulated by Con A. Thus HA and DS are much more stimulated by Con A and inhibited by NH4Cl than are CS and HS.