Biomedical subjects

J Doukas

Publications and source records attributed to J Doukas.

24 records · Page 2Linked to original sources

PubMed1990

Lymphocyte-mediated activation of cultured endothelial cells (EC). CD4+ T cells inhibit EC class II MHC expression despite secreting IFN-gamma and increasing EC class I MHC and intercellular adhesion molecule-1 expression.

Endothelial cells (EC) were cocultured with allogeneic PBL, CD4+ T cells, or CD8+ T cells, and the degrees of EC activation induced examined by determining patterns of endothelial class I and class II MHC and intercellular adhesion molecule-1 (ICAM-1) expression. Coculture with PBL or CD8+ T cells uniformly increases class I MHC and ICAM-1 expression on all EC within a culture, but induces class II MHC expression on only a subpopulation(s) of EC. This heterogeneous EC response to coculture contrasts with the uniform class II expression on all EC induced by IFN-gamma in replicate wells. CD4+ T cells, when compared to equal numbers of unfractionated PBL or CD8+ T cells, are more effective at increasing class I MHC and ICAM-1 but are unable to induce class II MHC expression. The failure of CD4+ T cells to induce EC class II MHC Ag is not due to insufficient activation of the T cells, as PHA-activated CD4+ T cells also do not induce significant class II expression. In addition, conditioned media (CM) from CD4+ T cell/EC contain greater levels of immunoreactive IFN-gamma than do CM from PBL/EC cocultures. Rather, CD4+ T cells appear to actively inhibit the induction of EC class II Ag but not class I or ICAM-1 by IFN-gamma. Inhibition occurs at the time of induction, as CD4+ T cells are not capable of down-regulating previously induced class II Ag. CM from CD4+/EC (but not PBL/EC) cocultures also inhibits IFN-gamma induction of EC class II MHC expression. The inhibitory activity is generated during CD4+ T cell-EC cell contact, and is enhanced by PHA. The inhibitory activity(ies) of the CD4+/EC-CM is as yet unidentified, and is only minimally reversible by cocktails of neutralizing antibodies directed against TNF-alpha, TNF-beta (lymphotoxin), IFN-alpha and IFN-beta. In conclusion, CD4+ and CD8+ T cells are each effective activators of EC, but the patterns of activation produced by these subsets are quite distinct, largely due to generation of a soluble inhibitor(s) of class II MHC induction during coculture of CD4+ T cells with EC.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1990

The potential roles of vascular endothelium in immune reactions.

Cell-mediated immune reactions are initiated and regulated by antigen specific CD4+ helper T cells. However, T cells cannot function independently. In order for a CD4+ T cell to recognize antigen, it must be presented in association with a class II major histocompatibility complex molecule by another cell type and, in order to lead to functional T-cell activation, the antigen presenting cell must also provide costimulatory signals. Once activated, CD4+ T cells function in vivo by secreting cytokines that elicit an inflammatory infiltrate of other cell types that serves to eliminate the source of foreign antigen. In vivo, the development of inflammation requires vascular responses as well as contributions of blood-derived leukocytes. Although several cell types in vitro can present antigen, provide costimulation, and perform actions that contribute to inflammation, vascular endothelial cells may be uniquely important immune accessory cells because they are anatomically uniquely positioned to function in vivo during cell-mediated immune reactions. In this report, we shall review recent data from our laboratories which further characterize the immune accessory functions of endothelial cells.

Antigen-Presenting Cells↗

PubMed1989

Vasoactive amines and eicosanoids interactively regulate both polymorphonuclear leukocyte diapedesis and albumin permeability in vitro.

Previously we reported that cultured endothelial cells (ECs) can promote or inhibit polymorphonuclear leukocyte (PMN) diapedesis and albumin permeability in vitro by altering monolayer intercellular integrity (an activity influenced by pretreatment with exogenous amines). Endothelial eicosanoid release was also seen to stimulate both PMN motility and diapedesis. We now demonstrate that these endothelial activities are related. Thromboxane (Tx) B2 pretreatment of ECs results in increased diapedesis and permeability across the monolayers whereas 6-keto-PGF1 alpha pretreatment has the opposite effects, demonstrating that these eicosanoids exert direct effects upon ECs, in addition to their direct effects upon PMNs as previously described. Norepinephrine (NE) or serotonin (5HT) pretreatment of ECs inhibits the release of TxB2 and 6-keto-PGF1 alpha, with the result that the stimulation of PMN motility by these EC metabolites is eliminated. In contrast, histamine increases the endothelial release of eicosanoids, resulting in a further increase in PMN motility. We conclude that histamine directly reduces EC monolayer integrity (by altering the endothelial cytoskeleton) and also increases eicosanoid release, actions which both enhance PMN motility and further reduce monolayer integrity. Conversely, NE and 5HT both increase intercellular integrity and decrease eicosanoid release, thereby decreasing PMN motility, diapedesis, and albumin permeability.

6-Ketoprostaglandin F1 alpha↗

PubMed1988

Endothelial-secreted arachidonic acid metabolites modulate polymorphonuclear leukocyte chemotaxis and diapedesis in vitro.

The influence of endothelial cells (ECs) on polymorphonuclear leukocyte (PMN) motility was examined using in vitro assays of PMN diapedesis and chemotaxis. ECs are seen to release arachidonic acid (20:4) metabolites that directly increase or decrease PMN movement, with their general effect being enhanced motility. This effect can be increased or decreased by treating ECs with stimulators or inhibitors of 20:4 metabolism, respectively. The metabolites include thromboxane B2, which increases PMN random motility, chemotaxis, and diapedesis in a dose-responsive manner and which acts as a chemoattractant; 6-keto-PGF1 alpha, which increases chemotaxis and diapedesis at high doses but decreases these responses at low doses; and a lipoxygenase-derived metabolite, suggested to be 5-hydroxyeicosatetraenoic acid, which increases chemotaxis and diapedesis. Thromboxane A2 and prostacyclin mimetics also affect chemotaxis in qualitatively similar manners as TxB2 and 6-keto-PGF1 alpha, respectively, but display greater potency. EC release of these metabolites is also seen to be substratum modulated, with an increased production by cells cultured on extracellular matrices. These results suggest that ECs are capable of modulating PMN motility and suggest a role for ECs in the control of PMN diapedesis.

Animals↗

PubMed1987

Vasoactive amines directly modify endothelial cells to affect polymorphonuclear leukocyte diapedesis in vitro.

Bovine aortic endothelial cells were cultured on the basement membrane surface of amnionic membrane and used as a substrate for polymorphonuclear leukocyte (PMN) diapedesis in vitro. Norepinephrine (NE), serotonin (5HT), or phalloidin treatment of the endothelial cells (ECs) reduces, whereas histamine or cytochalasin B increases, the number of PMNs migrating across the ECs and amnionic membrane. In contrast, amine treatment of PMNs or acellular amnionic membrane does not alter PMN diapedesis or chemotaxis. The NE and histamine effects are blocked by appropriate receptor antagonists, but the 5HT effect is not. All the agents' effects are also reversible. Qualitatively similar effects on EC permeability to Evan's blue-labeled albumin occur with all agents; however, PMN adhesion to ECs is not affected. Previously, we reported that NE and 5HT increase stress fiber numbers and decrease EC permeability to macromolecules in vitro, whereas histamine has the opposite effects, and that NE and 5HT eliminate the erythrocyte extravasation associated with thrombocytopenia in vivo. In this study, we propose that these vasoactive amines also alter PMN diapedesis in vitro through a direct effect on the EC, in part due to alterations in the EC cytoskeleton.

Animals↗

PubMedDate not supplied

Matrix-enabled gene transfer for cutaneous wound repair.

Several growth factor proteins have been evaluated as therapeutic agents for the treatment of chronic dermal wounds. Unfortunately, most have failed to produce significant improvements in wound healing, in part due to ineffective delivery and poor retention in the wound defect. It has been proposed that gene therapy might overcome the limitations of protein therapy via ongoing transcription and translation, thus prolonging the availability of the therapeutic protein. Reasoning that it would be of further benefit to ensure retention of the DNA vector as well as the therapeutic protein within the wound defect, we have evaluated matrix-enabled gene transfer for cutaneous wound repair (Gene Activated Matrix). Formulations consisting of bovine type I collagen mixed with adenoviral or plasmid gene vectors have been evaluated in 3 in vivo models. The therapeutic transgenes employed encode human platelet-derived growth factor-A or -B, proteins key to each phase of normal wound repair. Increased granulation tissue formation, vascularization, and reepithelialization have been shown compared to controls treated with collagen alone or collagen containing a reporter gene vector. Further enhancements of the tissue repair response have been achieved by combining matrix-enabled gene transfer with molecular targeting, in which the DNA vector is conjugated to a growth factor ligand (basic fibroblast growth factor). These promising results support the clinical evaluation of gene activated matrices for the treatment of chronic dermal wounds.

Animals↗