The immunopathology of experimental allergic encephalomyelitis. II. Endothelial cell Ia increases prior to inflammatory cell infiltration

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated neuroimmunologic disease model characterized by meningeal and parenchymal mononuclear cell infiltrates (see preceding companion paper). Here we report enhanced staining for Ia in the central nervous system (CNS) microvasculature endothelium in acute EAE in adult strain 13 guinea pigs (GP) sensitized with GP spinal cord homogenate (SC) or with GP myelin basic protein (MBP) in complete Freund's adjuvant (CFA). Cryostat sections of CNS and other tissues were stained with two monoclonal antibodies, 5S2 and 22C4, to GP Ia determinants, and with polyclonal antibody to factor VIII-related antigen (VIII-RA) as an endothelial cell marker. Morphometric techniques were employed on immunoperoxidase counterstained and coded sections to determine the frequency of Ia+ vessels and cells. Rare (approximately 10% of VIII-RA+) vascular endothelial cells were Ia+ in the CNS of normal and CFA-sensitized controls. SC- or MBP-sensitized strain 13 GP sacrificed on day 7, before the onset of neurologic signs (pre-clinical), had no detectable CNS mononuclear cell infiltrates, but had increased (approximately 30% of VIII-RA+) endothelial cell Ia staining over controls (p less than 0.001). The endothelial Ia staining persisted (approximately 35% of VIII-RA+) in vessels as the animals developed paralysis. There were no differences in endothelial cell Ia between SC- and MBP-induced disease. EAE-resistant strain 2 GP sensitized with SC/CFA had no neurologic signs, and had fewer inflammatory foci than strain 13 GP with EAE, but had similar numbers of Ia+ endothelial cells. No differences in endothelial cell Ia staining were found in non-CNS tissues among any GP groups. In EAE, increased endothelial cell Ia is a pre-inflammatory, target organ-specific alteration that persists during inflammation. The findings suggest that in vivo modulation of endothelial cell Ia may be part of the local immune response. Endothelial cells may play a significant role, in antigen presentation or in promoting T cell migration, in the in situ immune response in the CNS.     

Endothelial signaling in leukocyte transmigration

Leukocyte transendothelial migration is a multistep process coordinated by chemokine receptors, integrins and cell adhesion molecules. The interaction between leukocytes and endothelial cells is accompanied by bidirectional signaling in both cell types, which is initiated following formation of specialized, "docking" structures. In recent years, it has become clear that signaling in the endothelial cells importantly contributes to the transmigration process. This signaling induces a focal and transient loss of endothelial cell-cell adhesion, which is dependent on vascular-endothelial cadherin. Recent work from several groups has implicated Rho-like GTPases, reactive oxygen species, changes in the actin cytoskeleton and protein tyrosine phosphorylation in the control of VE-cadherin and associated proteins. This review discusses what is currently known about control of VE-cadherin function via intracellular signaling, and its induction by endothelial adhesion molecules of the immunoglobulin superfamily.     

Endothelial cell regulation by transforming growth factor-beta.

Pronounced changes including growth inhibition, increased matrix deposition and suppression of cell-associated proteolytic activity, take place in endothelial cells (EC) upon the application of TGF-beta. Interrelationships between these effects have shed some light on the mechanism of action of TGF-beta and on its role in regulating EC function vis-a-vis angiogenesis. For instance, preliminary evidence has indicated that increased levels of certain matrix components may be partly responsible for the antiproliferative action of TGF-beta. In addition, TGF-beta and bFGF have opposing effects on cellular proteolytic balance which may contribute to the antagonistic effect that TGF-beta has on bFGF-induced EC growth and possibly to the anti-angiogenic effect exerted by TGF-beta under certain circumstances. Of particular interest in this regard is the fact that physical contact between EC and vascular mural cells in EC:mural cell cocultures has been found to generate active TGF-beta, thus further implicating TGF-beta in the maintenance of the quiescent, differentiated aggregation of EC as found in vascular structures in vivo. While more information is needed to define what, if any role TGF-beta plays in endothelial differentiation, it is to be noted that many of the cellular and biochemical processes affected by TGF-beta are linked to differentiation. It is therefore possible that the growth inhibition of EC by TGF-beta primes them for differentiation and/or is critical for the maintenance of a differentiated state.     

Endothelial cell barrier regulation by sphingosine 1-phosphate

Disruption of vascular barrier integrity markedly increases permeability to fluid and solute and is the central pathophysiologic mechanism of many inflammatory disease processes, including sepsis and acute lung injury (ALI). Dynamic control of the endothelial barrier involves complex signaling to the endothelial cytoskeleton and to adhesion complexes between neighboring cells and between cells and the underlying matrix. Sphingosine 1-phosphate (S1P), a biologically active lipid generated by hydrolysis of membrane lipids in activated platelets, organizes actin into a strong cortical ring and strengthens both intercellular and cell-matrix adherence. The mechanisms by which S1P increases endothelial barrier integrity remain the focus of intense basic research. The downstream structural changes induced by S1P interact to decrease vascular permeability to fluid and solute, which translates into a reduction lung edema formation in animal models of ALI, thus suggesting a potentially life-saving therapeutic role for vascular barrier modulation in critically ill patients.     

Endothelial cell barriers: Transport of molecules between blood and tissues

An endothelial cell monolayer separates interstitia from blood and lymph, and determines the bidirectional transfer of solutes and macromolecules across these biological spaces. We review advances in transport modalities across these endothelial barriers. Glucose is a major fuel for the brain and peripheral tissues, and insulin acts on both central and peripheral tissues to promote whole‐body metabolic signalling and anabolic activity. Blood‐brain barrier endothelial cells display stringent tight junctions and lack pinocytic activity. Delivery of blood glucose and insulin to the brain occurs through their respective carrier (Glucose transporter 1) and receptor (insulin receptor), enacting bona fide transcytosis. At supraphysiological concentrations, insulin is also likely transferred by fluid phase cellular uptake and paracellular transport, especially in peripheral microvascular endothelia. The lymphatic microvasculature also transports insulin but in this case from tissues to lymph and therefrom to blood. This serves to end the hormone's action and to absorb highly concentrated subcutaneously injected insulin in diabetic individuals. The former function may involve receptor‐mediated transcytosis into lymphatic endothelial cells, the latter fluid phase uptake and paracellular transport. Lymphatic capillaries also mediate carrier‐dependent transport of other nutrients and macromolecules. These findings challenge the notion that lymphatic capillaries only transport macromolecules through intercellular flaps.     

Characterization of uterine leukocyte infiltration in gilts after artificial insemination

The objective of this study was to characterize the uterine leukocyte influx after artificial insemination (AI). After detection of oestrus with a boar at intervals of 1.5 h, seventy-two gilts were randomly assigned to a 2 x 3 x 4 factorial arrangement. AI was performed with 100 ml extended semen containing 5 x 10(9) spermatozoa (semen; n = 36) or 100 ml VSP semen extender (extender; n = 36) at one of three times after detection of oestrus: 12, 24 or 36 h (n = 24/time). The uterus was lavaged at 6, 12, 18 or 24 h (n = 18/time) after AI to determine the total number of uterine leukocytes. In addition, uterine lavage was performed on nine untreated gilts immediately after the detection of oestrus to establish a baseline number of leukocytes. The leukocyte response in all samples consisted predominately (92-99%) of polymorphonuclear neutrophilic granulocytes (PMNs). The mean number of PMNs recovered from the uteri of gilts treated with semen was greater than in gilts treated with extender and in untreated gilts (P < 0.01). The greatest number of PMNs in semen-treated gilts was found 12 h after AI (P < 0.01), and this number was sustained for 24 h. In contrast, the number of uterine PMNs recovered from extender-treated gilts reached a peak at 6 h and had declined by 12 h after AI (P < 0.05). It was concluded that an extensive influx of PMNs into the uterus is a normal sequence to AI. The consequences and importance of semen-induced uterine leukocytosis needs further investigation.     

Dynamic regulation of activated leukocyte cell adhesion molecule-mediated homotypic cell adhesion through the actin cytoskeleton

Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM-ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM-ALCAM interactions. This induction of cell adhesion is likely due to clustering of ALCAM at the cell surface, which is observed after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after CytD treatment. In contrast, both surface distribution and adhesion of a glycosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to CytD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatment. This demonstrates that clustering of ALCAM is essential for cell adhesion, whereas enhanced diffusion of ALCAM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the freely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduced over time, suggesting strengthening of the cytoskeleton linkage. From these findings we conclude that activation of ALCAM-mediated adhesion is dynamically regulated through actin cytoskeleton-dependent clustering.     

PDX regulates inflammatory cell infiltration via resident macrophage in LPS‐induced lung injury

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti‐inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT‐PCR, and ELISA was used to measure MIP‐2, MCP‐1, TNF‐α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 10⁶) were cultured with 1 μg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS‐induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP‐1, MIP‐2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF‐α/MIP‐2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS‐stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.     

Cytosolic phospholipase A2-mediated regulation of phospholipase D2 in leukocyte cell lines.

Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst. Two distinct PLD isoforms, designated PLD1 and PLD2, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear. In our present study we investigated how PLD2 activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD2, and in PLD2 -transfected COS-7 cells. Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lines, suggesting that Ca2+ might be implicated in the regulation of the PLD2 activity. In addition to the A23187-induced PLD2 activation, A23187 also increases PLA2-mediated arachidonic acid release, and the A23187-stimulated PLD2 and PLA2 activities could be blocked by pretreatment of the cells with cytosolic calcium-dependent PLA2 (cPLA2) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induced PLD2 and PLA2 activities could be inhibited by cotransfection with antisense cPLA2 oligonucleotide. These results suggest a role for cPLA2 in the regulation of PLD2 activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD2 activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD2 activity is up-regulated by Ca2+ influx and that cPLA2 may play a key role in the Ca2+-dependent regulation of PLD2 through generation of lysophosphatidylcholine.     

Vatairea macrocarpa lectin induces paw edema with leukocyte infiltration

A lectin from Vatairea macrocarpa (Vmac) seeds was investigated in a model of paw edema in rats and the possible involvement of leukocytes. Vmac (200 and 400 microg/paw, s.c.) induced a significant time- and dose-dependent paw edema, with leukocyte infiltration, which was drastically reduced in leukopaenic animals. These data suggest a pro-inflammatory effect for this lectin that is dependent on the presence of leukocytes.     

Fas ligand overexpression on allograft endothelium inhibits inflammatory cell infiltration and transplant-associated intimal hyperplasia

Despite recent advances in immunosuppressive therapy, accelerated coronary atherosclerosis remains a major problem in the long-term survival of transplant recipients. Chronic graft vasculopathy is believed to result from recipient inflammatory responses, and it is characterized by early mononuclear cell infiltration of the transplanted vessel. Here we show that endothelial cells can be genetically modified to overexpress functional, cell-surface Fas ligand (FasL) by adenovirus-mediated gene transfer without undergoing self-destruction. In a rodent model of transplant graft vasculopathy, endothelial overexpression of FasL attenuated T cell and macrophage infiltration at 1 wk posttransplantation. These vessels also displayed reduced neointima formation at one and 2 mo posttransplantation. These results indicate that inhibition of the early inflammatory response to allografted vessels by endothelial cell-specific overexpression of FasL may have utility in the treatment of transplant arteriosclerosis.