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J R Gamble

Publications and source records attributed to J R Gamble.

At least 55 records · Page 3Linked to original sources
PubMed1994

The role of granulocyte-macrophage and granulocyte colony-stimulating factors in neutrophil transendothelial migration: comparison with interleukin-8.

Granulocyte-macrophage and granulocyte colony-stimulating factors (GM-CSF and G-CSF) have proinflammatory effects on mature neutrophils. Both factors have been reported to cause neutrophil chemotaxis and are produced by cells stimulated by inflammatory mediators, including endothelial cells. We therefore tested the hypothesis that these factors might mediate neutrophil transendothelial migration, either by forming a gradient across the endothelial monolayer or through the production of CSFs by activated endothelium. Studies of neutrophil migration across filters without endothelium showed that migration was promoted in the presence of a gradient of either CSF, but was equally promoted against the gradient; that is, the CSFs are chemokinetic but not chemotactic. The CSFs promoted migration of neutrophils across endothelial monolayers cultured on filters, but the magnitude of this effect was very small compared with that of a prototypic neutrophil chemoattractant, interleukin-8 (IL-8) (migration index [stimulated/unstimulated] 1.8-fold for GM-CSF, 10.8-fold for IL-8). Activation of endothelial monolayers by preincubation with tumor necrosis factor-alpha (TNF-alpha) increased neutrophil transmigration significantly; neutralizing antibodies to IL-8 inhibited this increase by 44%, whereas neutralizing anti-GM-CSF antibodies did not inhibit it. These data suggest little role for the CSFs in neutrophil diapedesis at inflammatory sites in vivo. Exposure of neutrophils to GM-CSF decreased their migration through TNF-activated monolayers, whereas G-CSF did not. This may have implications for the therapeutic administration of these factors.

Antibodies↗
PubMed1994

Characterization of a spontaneously transformed human endothelial cell line.

BACKGROUND: A line of cells was isolated from a focus observed in a human umbilical vein endothelial cell (HUVEC) culture, presumably the result of a spontaneous transformation event. EXPERIMENTAL DESIGN: The cell line was continuously cultured for 16 months, after which time, the proliferative rate, capacity to be cloned, and ability to be transfected was investigated. The cell line was analyzed for expression of endothelial cell markers, von Willebrand Factor, P selectin, and scavenger receptor. We also examined the tumor necrosis factor (TNF)-mediated upregulation of E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. We evaluated the levels of expression and types of TNF-alpha receptor expressed by the cell line, and the cell lines response to interleukin-4 and interferon-gamma. CD34 expression of this cell line and the ability of transforming growth factor-beta to inhibit the TNF-alpha induction of E-selectin was examined. The ability to support neutrophil adhesion and transmigration, and generate capillary-like tubes in vitro was assessed. Finally, the karyotype and tumourigenicity of this line was established. RESULTS: This cell line (C11STH) has a doubling time comparable to that of normal HUVECs and has a trisomy of chromosome 8 and 11. The cell line is capable of generating colonies at clonal density, and is transfectable with efficiencies comparable to normal HUVECs. C11STH expresses von Willebrand factor, P-selectin, and scavenger receptor to an extent similar to passaged HUVECs and can be induced to express E-selectin, VCAM, and ICAM with TNF-alpha. C11STH expresses both the p55 and p75 subunits of TNF-alpha receptor at levels similar to HUVECs. The ability of interleukin-4 to enhance the expression of VCAM and reduce the TNF-alpha-mediated expression of E-selectin is maintained in this cell line. C11STH cells are unable to induce class II major histocompatibility antigen in response to interferon-gamma. However, interferon-gamma is able to synergize with TNF to enhance the expression of E-selectin. C11STH cells do not express CD34 or show transforming growth factor-beta inhibition of TNF-alpha induced E-selectin expression, functions indicative of primary, or early passage HUVECs. The cell line retains the ability to support neutrophil adhesion and transmigration and can generate patent tubes when seeded onto complex basement membrane gels. However, the cell line no longer has the ability to generate capillary-like vessels when seeded onto collagen gels in the presence of phorbol 12-myristate 13-acetate. CONCLUSIONS: C11STH has retained many of the normal characteristics of endothelial cells, is able to proliferate at clonal density, and is easily transfectable. The line will provide a useful resource for endothelial cell biology. Its ability to make capillary-like tubes on basement membrane gels, but not on collagen will provide a powerful tool with which to further analyze the processes involved in angiogenesis, and will enable us to define the role of specific proteins in angiogenesis. Since C11STH shows tube competence on Matrigel, but is not tube competent on collagen, our studies suggest that capillary-tube formation on Matrigel and collagen occur via qualitatively different mechanisms. Thus, this cell line provides the opportunity to examine the signalling mechanisms required to generate capillary tube formation. These may include the involvement of matrix molecules, the production of proteases and inhibitors, gene regulation and kinases or phosphatases.

Cell Adhesion↗
PubMed1993

The receptor for interleukin 3 is selectively induced in human endothelial cells by tumor necrosis factor alpha and potentiates interleukin 8 secretion and neutrophil transmigration.

Interleukin (IL)-3 stimulates hemopoiesis in vitro. However, IL-3 is not normally found in bone marrow, raising doubts as to the in vivo role of IL-3. We have found that human umbilical vein endothelial cells (HUVEC) express functional high-affinity receptors for IL-3 after stimulation with tumor necrosis factor alpha (TNF-alpha), IL-1 beta, or lipopolysaccharide, and that this receptor is involved in inflammatory phenomena. TNF-alpha caused time- and dose-dependent up-regulation of mRNA for the IL-3 receptor alpha and beta chains, with maximal effects occurring 16-36 h after stimulation with TNF-alpha at 100 units/ml. Induction of mRNA correlated with protein expression on the cell surface as judged by monoclonal antibody staining and by the ability of HUVEC to specifically bind 125I-labeled IL-3. Scatchard analysis under optimal conditions of TNF-alpha stimulation revealed approximately 1500 IL-3 receptors per cell, which were of a high-affinity class (Kd = 500 pM) only. In contrast to a previous report, receptors for granulocyte-macrophage colony-stimulating factor could not be detected. IL-3 binding to TNF-alpha-activated HUVEC enhanced IL-8 production, E-selection expression, and neutrophil transmigration. The selective induction of a functional IL-3 receptor on endothelial cells suggests that, beyond hemopoiesis, IL-3 may have an important role in chronic inflammation and in allergic diseases.

Cell Adhesion Molecules↗
PubMed1993

Transforming growth factor-beta inhibits E-selectin expression on human endothelial cells.

Transforming growth factor-beta (TGF-beta), a pleiotropic cytokine that is elaborated in the active form upon co-culture of endothelial cells and pericytes or smooth muscle cells, has been shown to decrease the adhesiveness of endothelial cells for neutrophils, lymphocytes, and tumor cells. The mechanism whereby TGF-beta inhibits the adhesiveness of human endothelial cells was investigated. TGF-beta inhibited the basal E-selectin (formerly ELAM-1) expression by 55 +/- 7% and TNF-stimulated expression by 57 +/- 4%. Similar decreases of IL-1-stimulated expression were also seen. Peak inhibition was seen at TGF-beta doses between 0.2 and 2 ng/ml. Both TGF-beta 1 and -beta 2 were functional. The effectiveness of TGF-beta in inhibiting E-selectin expression was dependent on cell density and incubation time. TGF-beta also inhibited E-selectin mRNA levels in endothelial cells. TGF-beta had no effect on the expression of VCAM-1 and ICAM-1, but was additive with IL-4 in inhibiting the expression of E-selectin. The expression of E-selectin has been shown to mediate several aspects of the inflammatory response involving neutrophils and memory T lymphocytes. Perivascular TGF-beta appears to act as an inhibitor of the expression of the endothelium-specific selectin, E-selectin, and therefore of inflammatory responses involving neutrophils and (a subset of) lymphocytes.

Base Sequence↗
PubMed1993

Regulation of in vitro capillary tube formation by anti-integrin antibodies.

Human endothelial cells are induced to form an anastomosing network of capillary tubes on a gel of collagen I in the presence of PMA. We show here that the addition of mAbs, AK7, or RMAC11 directed to the alpha chain of the major collagen receptor on endothelial cells, the integrin alpha 2 beta 1, enhance the number, length, and width of capillary tubes formed by endothelial cells derived from umbilical vein or neonatal foreskins. The anti-alpha 2 beta 1 antibodies maintained the endothelial cells in a rounded morphology and inhibited both their attachment to and proliferation on collagen but not on fibronectin, laminin, or gelatin matrices. Furthermore, RMAC11 promoted tube formation in collagen gels of increased density which in the absence of RMAC11 did not allow tube formation. Neither RMAC11 or AK7 enhanced capillary formation in the absence of PMA. Lumen structure and size were also altered by antibody RMAC11. In the absence of antibody the majority of lumina were formed intracellularly from single cells, but in the presence of RMAC11, multiple cells were involved and the lumen size was correspondingly increased. Endothelial cells were also induced to undergo capillary formation in fibrin gels after PMA stimulation. The addition of anti-alpha v beta 3 antibodies promoted tube formation in fibrin gels and inhibited EC adhesion to and proliferation on a fibrinogen matrix. The enhancement of capillary formation by the anti-integrin antibodies was matrix specific; that is, anti-alpha v beta 3 antibodies only enhanced tube formation on fibrin gels and not on collagen gels while anti-alpha v beta 1 antibodies only enhanced tubes on collagen and not on fibrin gels. Thus we postulate that changes in the adhesive nature of endothelial cells for their extracellular matrix can profoundly effect their function. Anti-integrin antibodies which inhibit cell-matrix interactions convert endothelial cells from a proliferative phenotype towards differentiation which results in enhanced capillary tube formation.

Animals↗
PubMed1993

Chemotactic desensitization of neutrophils demonstrates interleukin-8 (IL-8)-dependent and IL-8-independent mechanisms of transmigration through cytokine-activated endothelium.

We have recently shown that an exogenous gradient of interleukin-8 (IL-8) induces the transendothelial migration of neutrophils. Treatment of endothelium with the cytokines IL-1 or tumour necrosis factor (TNF) also causes neutrophil transmigration, and recent evidence suggests that this may be due to endogenous IL-8 produced by the endothelium. We have used specific chemotactic desensitization of neutrophils to investigate the role of IL-8 in transmigration through cytokine-activated endothelium. Preincubation of neutrophils with IL-8 reduced their chemotactic transmigration response to an IL-8 gradient by 81%, demonstrating desensitization. Transmigration in response to cytokine-activated endothelium was inhibited by 104% after IL-8 preincubation, thus tending to support the role of IL-8. However, preincubation with another neutrophil chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (FMLP), which did not affect the IL-8, response, also inhibited transmigration, by 74%. This suggests that FMLP preincubation acts to inhibit a non-IL-8-dependent mechanism of transmigration through cytokine-activated endothelium. Chemotactic factor pretreatment of neutrophils did not reduce their adhesion to activated endothelium, but specifically blocked the transmigration step. We have therefore shown that chemotactic transmigration can be subjected to factor-specific desensitization, and have used this to provide evidence supporting a role for IL-8 in transmigration through cytokine-activated endothelium, as well as suggesting a further IL-8-independent mechanism. These data also provide a mechanism for the observed defect in accumulation of neutrophils at inflammatory sites when chemotactic factors are infused intravenously.

Cells, Cultured↗
PubMed1992

Characterization of GMP-140 (P-selectin) as a circulating plasma protein.

GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.

Amino Acid Sequence↗
PubMed1991

Inhibition of human monocyte adhesion by interleukin-4.

The adhesion of monocytes to vascular surfaces is central to inflammation and atherogenesis; however, very little is known about regulatory factors that can prevent these processes. Here we report the inhibition of human monocyte adhesion to human endothelial layers and plastic by interleukin-4 (IL-4), a T-cell-derived glycoprotein with pleiotropic activities. The inhibitory effects of IL-4 were seen with basal and cytokine-stimulated monocyte adhesion, were apparent at low concentration, and were abolished by inactivating IL-4. No direct toxic effect of IL-4 on monocytes was detected. Inhibition of adhesion was accompanied by small increases in monocyte surface expression of the leukocyte-functional antigen group of adhesion structures, suggesting that absolute levels of expression may be less important than the functional status of such molecules in the regulation of monocyte adhesion. In addition, inhibition by IL-4 of cytokine-stimulated monocyte adhesion was not associated with changes in the surface expression of cytokine receptors. These results suggest a role for IL-4 in the regulation of monocyte adhesion, and may provide for a common mechanism for the inhibitory effects of IL-4 on monocyte function.

Antigens, CD↗
PubMed1991

Adhesion protein GMP140 inhibits superoxide anion release by human neutrophils.

The respiratory burst of blood neutrophils has a critical role in the destruction of microorganisms and tissue damage in inflammation. Neutrophils adhere in a dose-dependent fashion to granule membrane protein 140 (GMP140), a member of the LEC-CAM (lectin/epidermal growth factor/complement-binding domain cell adhesion molecule) family of adhesion proteins when it is immobilized onto plastic surfaces. Adherence to GMP140 was associated with less superoxide anion generation than adherence to other surfaces, an effect that is especially remarkable after activation of neutrophils with tumor necrosis factor alpha, an agent that on other surfaces promotes adhesion and spreading. However, on GMP140 the cells fail to spread and instead remain rounded and refractile. Neutrophils adhering to GMP140 were also deficient in superoxide anion generation to formylmethionylleucylphenylalanine. Furthermore, fluid-phase GMP140 also inhibited the superoxide generation by neutrophils stimulated by tumor necrosis factor alpha. The effect of GMP140 was reversible by washing and was inhibited by anti-GMP140 Fab antibody. GMP140 appears to be a natural antiinflammatory molecule that may prevent the inappropriate activation of neutrophils in the circulation.

Antibodies↗
PubMed1991

Endothelial cell adhesiveness for human T lymphocytes is inhibited by transforming growth factor-beta 1.

Recombinant human transforming growth factor-beta (TGF-beta) was found to inhibit the adhesive phenotype of human umbilical vein endothelial cells for human PBL, purified T lymphocytes, and PHA-activated lymphoblasts. TGF-beta inhibited lymphocyte attachment to resting human umbilical vein endothelial cells and also to endothelial monolayers stimulated with the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Our investigations also show that the ability of endothelial cells to respond to TGF-beta by altering their adhesiveness is lost with prolonged culture of the cells. However, this loss is selective as TGF-beta inhibits cell proliferation in both early and late passage endothelial cells. These results suggest that in vivo TGF-beta may inhibit the adhesive phenotype of endothelial cells and also may limit the immunologic response occurring at the endothelial cell barrier.

Cell Adhesion↗
PubMed1991

Interleukin-8 induces neutrophil transendothelial migration.

Interleukin-8 (IL-8) is a potent neutrophil chemotactic stimulant. We have used chemically synthesized IL-8 to investigate its role in human neutrophil adhesion and transendothelial migration. IL-8 enhanced the adhesiveness of human neutrophils to plastic, and to both unstimulated and tumour necrosis factor (TNF)-stimulated endothelial monolayers in vitro. Using a two-compartment model separated by a confluent endothelial monolayer, we have shown that IL-8 chemotactic stimulation induced transmigration across the monolayer of up to 87.4 +/- 2.1% of added neutrophils (compared to random unstimulated transmigration of 2.2 +/- 0.7%), while chemokinetic stimulation led to transmigration of 21 +/- 3.8% of neutrophils. Preincubation of endothelium with TNF also induced transmigration in this model, and was additive when combined with an IL-8 chemotactic stimulus. Endothelial permeability was increased at maximal rates of chemotactic transmigration, which may correlate with increased permeability of vessels at inflammatory sites in vivo. The property of IL-8 to stimulate movement of neutrophils across endothelial monolayers in vitro supports the concept of a central role for this molecule in the accumulation of neutrophils at inflammatory lesions in vivo.

Capillary Permeability↗
PubMed1990

Prevention of activated neutrophil adhesion to endothelium by soluble adhesion protein GMP140.

Neutrophils and monocytes, but not lymphocytes, adhered strongly to plastic surfaces coated with GMP140, a protein of endothelial cells and platelets. This adhesion of neutrophils was mediated by GMP140 and not by the CD18 integrin complex. By contrast, GMP140 in solution inhibited the CD18-dependent adhesion of tumor necrosis factor-alpha-activated neutrophils to plastic surfaces and resting endothelium, but not of resting neutrophils to tumor necrosis factor-alpha-activated endothelium. Thus, the binding of a soluble form of an adhesion protein selectively inhibited another set of adhesive events. Soluble GMP140 may be important in maintaining the nonadhesiveness of neutrophils in the circulation and may serve to limit inflammatory reactions.

Antibodies↗
PubMed1990

IL-3 and granulocyte-macrophage colony-stimulating factor stimulate two distinct phases of adhesion in human monocytes.

IL-3 and granulocyte-macrophage CSF are hemopoietic growth factors involved in monocytopoiesis and functional stimulation of circulating blood monocytes. We demonstrate that both cytokines enhance the adhesion of purified human monocytes to cultured human umbilical vein endothelial cells and to plastic surfaces. The stimulation seen was biphasic: an early phase detectable by 10 min, and a late phase seen after 9 h of in vitro culture. IL-3- and granulocyte-macrophage-CSF-stimulated adhesion was seen at concentrations as low as 6 pM, with maximal monocyte adhesion of up to 60% seen at concentrations of 60 pM and above. Both phases of stimulated adhesion were partially inhibited by a monoclonal antibody to CD18, the common beta-chain of the leukocyte functional Ag family of adhesion molecules, but not by an antibody to CD11b, the alpha-chain of MAC-1. However, a difference in the mechanism by which the early and late phases of stimulated adhesion arise could be shown by the use of cycloheximide as an inhibitor of protein synthesis. Although the late phase was totally dependent on de novo protein synthesis, early phase adhesion was not inhibited by cycloheximide, suggesting receptor redistribution or conformational change as the mechanism mediating enhanced adhesion at this time. These findings may be relevant to the pathogenesis of inflammatory disease and may have implications for the clinical use of these cytokines.

Antibodies, Monoclonal↗
PubMed1990

Heterogeneity of recombinant granulocyte-macrophage colony-stimulating factor-mediated enhancement of neutrophil adherence to endothelium.

Human recombinant (r) and chemically synthesized granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to enhance the attachment of neutrophils to monolayers of human umbilical vein endothelial cells by direct action upon the neutrophil. Using synthetic peptides of GM-CSF with truncated amino and carboxy termini, a region between amino acids 14 and 24 was found to be essential for neutrophil attachment. In analysis of the response of neutrophils from individual donors, a heterogeneity in their capacity to respond to GM-CSF by increased adherence was observed. The level of response to GM-CSF did not depend on receptor number. However, a positive correlation (r = 0.58) was found between the ability to respond to GM-CSF and the level of response to tumor necrosis factor--suggesting a link between the responses of neutrophils to these two cytokines. The stimulation of neutrophil adhesiveness to endothelial cells by rGM-CSF and the heterogeneity in donor response may have important implications for the clinical administration of GM-CSF.

Adult↗