Dorian O. Haskard

Detection of a circulating form of vascular cell adhesion molecule-1: raised levels in rheumatoid arthritis and systemic lupus erythematosus.

We have developed a panel of MoAbs against four separate but overlapping epitopes on endothclial cell (EC) vascular cell adhesion molecule‐1 (VCAM‐1). Two of the MoAbs(1G11 and IE5) inhibited T cell adhesion to tumour necrosis factor (TNF)‐activated EC, whilst two MoAbs (1.4C3 and 6D9) did not. Using these MoAbs we have identified a circulating form of VCAM‐1 (cVCAM‐l) which has identical epitope distribution to the EC form, and which is able to support the adhesion of the human lymphoblastoid cell line Jurkat J6 by a VLA‐4‐ and VCAM‐1‐dependent mechanism when immobilized from plasma. cVCAM‐l isolated by immunoaffinity and size‐exclusion chromatographies was shown by SDS‐PAGE to have an apparent mol. wt of 85 90 kD. Levels of cVCAM‐l were significantly raised (P < 0.001) in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) compared with normal individuals. It is possible that cVCAM‐l may be a useful plasma marker for the diagnosis and management of patients with inflammatory diseases. Furthermore, detection of elevated cVCAM‐l levels may act as a guide to the importance of VCAM‐1‐dependent cell adhesion in different pathological settings.

Leucocyte Endothelial Cell Adhesion: a Study comparing Human Umbilical Vein Endothelial Cells and the Endothelial Cell Line EA‐hy‐926

EA‐hy‐926 is a cell line produced by hybridizing human umbilical vein endothelial cells (HUVEC) and the epithelial cell line A549. To establish whether EA‐hy‐926 could be used as a model for endothelial cells (EC) in leucocyte‐EC adhesion interactions, the effect of interleukin‐4 (IL‐4), tumour necrosis factor (TNF) or interferon‐γ (IFN) stimulation on their adhesiveness and expression of E‐selectin, vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1) was compared with that of HUVEC and A549. Although HUVEC exhibited increased adhesiveness and adhesion molecule expression with IL‐4, TNF or IFN, EA‐hy‐926 exhibited these responses only with TNF. CD11/CD 18‐dependent binding accounted for a significant component of basal binding to HUVEC and EA‐hy‐926, but did not account for the increased binding of T cells, JY, J6, ICH‐BJ or ICH‐KM cell lines to TNF‐stimulated monolayers. At least part of the CD1l/CD18‐independent adhesion was attributable to VCAM‐1 induction on HUVFC and FA‐hy‐926. TNF‐stimulation also induced F‐selectin expression on EA‐hy‐926 and HUVEC and an accompanying increase in neutrophil (PMN) binding. The EA‐hy‐926 cells used in this study, therefore, showed responses similar to HUVEC when stimulated with TNF but not when stimulated with IL‐4 or IFN.

Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties

Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions.

Induction of endothelial cell decay-accelerating factor by vascular endothelial growth factor: a mechanism for cytoprotection against complement-mediated injury during inflammatory angiogenesis.

OBJECTIVEnDecay-accelerating factor (DAF) is a widely expressed, multifunctional cell surface protein involved in complement regulation and cell signaling. Previous studies have demonstrated that endothelial cell (EC) DAF is up-regulated by tumor necrosis factor alpha and inhibits complement binding. Because vascular endothelial growth factor (VEGF) is cytoprotective to endothelium and is expressed at sites of chronic inflammation, we hypothesized that VEGF may induce DAF expression during inflammatory angiogenesis.nnnMETHODSnHuman umbilical vein and dermal microvascular EC were isolated using routine procedures, and the regulation and function of DAF, as well as other complement-regulatory proteins (membrane cofactor protein and CD59), were analyzed following stimulation with VEGF.nnnRESULTSnIncubation of large- or small-vessel EC with VEGF led to increased expression of DAF, with maximal expression after 48-72 hours of stimulation. This effect depended on the activation of protein kinase C (PKC) and required increased steady-state messenger RNA levels and de novo protein synthesis. Although VEGF-induced EC proliferation was inhibited by both p38 and p42/44 mitogen-activated protein kinase (MAPK) antagonists, DAF up-regulation in response to VEGF was only sensitive to inhibition of p38 MAPK. VEGF-stimulated EC showed a 60% reduction in C3 deposition following complement activation, and this resulted in a marked reduction in complement-mediated EC lysis. These protective effects were abolished by anti-DAF monoclonal antibody 1H4.nnnCONCLUSIONnThis study confirms the importance of PKC for the regulation of DAF expression by EC and reveals VEGF to be a physiologic agonist for this pathway. The up-regulation of DAF expression by VEGF may represent an important mechanism for the protection of EC from complement-mediated injury during angiogenesis in inflammatory rheumatic diseases.

Human umbilical vein and dermal microvascular endothelial cells show heterogeneity in response to PKC activation

Changes in endothelial cell (EC) phenotype are central to the function of endothelium in inflammation. Although these events mainly occur in the microvasculature, previous studies have predominantly used large-vessel EC. Using enzyme-linked immunosorbent and flow cytometric assays, we compared the responses of human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (DMEC) to the activation of protein kinase C (PKC). Stimulation with phorbol 12,13-dibutyrate and more selective PKC agonists, including 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), induced morphological changes and proliferation in both EC types. PKC activation induced a marked increase in Thy-1 expression on DMEC and only a moderate rise on HUVEC. Furthermore, heterogeneity in the induction of the adhesion molecules intercellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin between the two EC types following activation of PKC was demonstrated. In particular, E-selectin and VCAM-1 were significantly upregulated on HUVEC but not DMEC. The data indicate that the PKC pathway is unlikely to be important for E-selectin and VCAM-1 expression in the microvasculature but are consistent with a role for PKC in angiogenesis. This diversity in signaling in response to PKC activation may depend on differential utilization of PKC isozymes and may facilitate specialized endothelial responses.Changes in endothelial cell (EC) phenotype are central to the function of endothelium in inflammation. Although these events mainly occur in the microvasculature, previous studies have predominantly used large-vessel EC. Using enzyme-linked immunosorbent and flow cytometric assays, we compared the responses of human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (DMEC) to the activation of protein kinase C (PKC). Stimulation with phorbol 12,13-dibutyrate and more selective PKC agonists, including 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), induced morphological changes and proliferation in both EC types. PKC activation induced a marked increase in Thy-1 expression on DMEC and only a moderate rise on HUVEC. Furthermore, heterogeneity in the induction of the adhesion molecules intercellular adhesion molecule 1, vascular cell adhesion molecule 1 IVCAM-1), and E-selectin between the two EC types following activation of PKC was demonstrated. In particular, E-selectin and VCAM-1 were significantly upregulated on HUVEC but not DMEC. The data indicate that the PKC pathway is unlikely to be important for E-selectin and VCAM-1 expression in the microvasculature but are consistent with a role for PKC in angiogenesis. This diversity in signaling in response to PKC activation may depend on differential utilization of PKC isozymes and may facilitate specialized endothelial responses.

Stromal Cell-Derived Factor 1 (CXCL12) Induces Human Cell Migration into Human Lymph Nodes Transplanted into SCID Mice

Stromal cell-derived factor 1 (SDF-1; CXCL12), a CXC chemokine, has a primary role in signaling the recruitment of hemopoietic stem cell precursors to the bone marrow during embryonic development. In postnatal life, SDF-1 is widely expressed and is induced in chronically inflamed tissues such as psoriatic skin and the rheumatoid synovium, but has also been implicated in the migration of lymphocytes to lymphoid organs. To investigate the role of SDF-1 in recirculation and homing in vivo, we have developed a model in which human peripheral lymph nodes (huPLN) are transplanted into SCID mice. We have shown that huPLN transplants are viable, vascularized by the murine circulation that forms functional anastomoses with transplant vessels. In addition, grafts retain some features of the pretransplantation tissue, such as lymphoid follicles, lymphatic and high endothelial venule markers. We also show that SDF-1 is capable of inducing the migration of a SDF-1-responsive cell line (U937) and human PBLs from the murine circulation into the grafts in a dose-dependant manner, inhibitable by CXCR4 blockade. The mechanism of action of SDF-1 in this model is independent from that of TNF-α and does not rely on the up-regulation of adhesion molecules (such as ICAM-1) on the graft vascular endothelium. This is the first description of huPLN transplantation into SCID mice and of the functional effects of SDF-1 in regard to the migration of human cells into huPLN in vivo. This model provides a powerful tool to investigate the pathways involved in cell migration into lymphoid organs and potentially to target them for therapeutic purposes.

Targeting endothelium for gene therapy via receptors up-regulated during angiogenesis and inflammation.

Abstractu2003Endothelial cells are an attractive target for gene therapy because they are intimately involved in disease processes associated with inflammation and angiogenesis and because endothelial cells are readily accessible to gene therapy vectors via the circulation. Furthermore, specific receptors are up-regulated during angiogenesis or during inflammation. Therefore it should be possible to target the specific populations of endothelial cells involved in each of these disease processes. We have utilized two bispecific antibodies to target entry of an adenovirus vector into endothelial cells expressing a receptor up-regulated during angiogenesis (αv integrins) and a receptor up-regulated during inflammation (E-selectin). Both bispecific antibodies contain the anti-FLAG M2 mAb, which binds to a FLAG epitope genetically incorporated into the penton base protein of the vector, AdFLAG. The anti-αv-integrin×anti-FLAG bsAb was able to direct binding and entry of AdFLAG into endothelial cells via αv integrins. Likewise, the anti-E-selectin×anti-FLAG bsAb was able to direct binding and entry of AdFLAG into tumor-necrosis-factor(TNF)-activated endothelial cells via E-selectin. Endothelial cells not activated with TNF were not efficiently transduced by the AdFLAG/E-selectin bsAb complex. These results demonstrate that bispecific antibodies can be successfully used to target adenovirus to endothelially expressed receptors that are up-regulated during angiogenesis or inflammation.

Measurement of mRNA for E-selectin, VCAM-1 and ICAM-1 by reverse transcription and the polymerase chain reaction

Stimulation of cultured human umbilical vein endothelial cells by cytokines such as interleukin-1 and tumour necrosis factor induces de novo synthesis and expression of the adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In general, alterations in cell surface expression of these molecules are known to be related to increased gene transcription and altered levels of mRNA. The extension of these observations to the study of inflammatory processes in different human organs necessitates the development of techniques for the quantification of mRNA in small tissue samples. Here we present a method for the quantification of mRNA for E-selectin, VCAM-1 and ICAM-1 using reverse transcription and the polymerase chain reaction (RT-PCR). For each molecule of interest a mutant RNA was synthesised consisting of the wild-type sequence deleted of 15-20 bases. The mutant and wild-type RNA sequences are recognised by the same primers, and can therefore be amplified competitively in the same tube by RT-PCR. As the mutant and wild-type RNAs compete for the primers, the amount of wild-type RNA can be determined by the size of the dominant product that results after addition of known quantities of mutant RNA. Using this detection and quantification method we have examined the dose dependency and time course of mRNA accumulation following TNF-alpha stimulation of HUVEC. Similar time-courses of E-selectin, ICAM-1 and VCAM-1 mRNA accumulation were observed by competitive RT-PCR as by laser densitometry of Northern blots. Finally we were able to show that the technique could measure changes in levels of mRNA for these three molecules in human skin biopsies taken at different times during the development of a delayed hypersensitivity response to tuberculin purified protein derivative. This technique should be useful for the study of adhesion molecule mRNA in small tissue culture samples and in biopsies.

Enhanced Recruitment of Th2 and CLA-Negative Lymphocytes by the S128R Polymorphism of E-Selectin

E-selectin is a cytokine-inducible endothelial cell adhesion molecule that binds a restricted population of T lymphocytes consisting of Th1 memory cells bearing the cutaneous lymphocyte Ag (CLA). A serine to arginine (S128R) polymorphism in E-selectin has been reported at increased frequency in patients with systemic lupus erythematosus and atherosclerosis. Here we tested the hypothesis that the S128R substitution may contribute to increased vascular disease by altering the number and/or phenotype of lymphocytes interacting with E-selectin under shear flow. We observed that CHO cell monolayers transfected with S128R recruited significantly greater numbers of unfractionated lymphocytes than monolayers expressing an equivalent density of wild-type (WT) E-selectin. Depletion of the CLA+ subpopulation or generation of CLA− lymphoblasts abolished rolling and arrest on WT E-selectin, but left a residual population that interacted with S128R. Generation of Th subsets revealed preferential interaction of Th0 and Th2, but not Th1, cells with S128R compared with WT. However, only T cells of a memory phenotype interacted with S128R, since neither monolayer supported rolling of CD45RA+ cells. Our results demonstrate that the S128R polymorphism extends the range of lymphocytes recruited by E-selectin, which may provide a mechanistic link between this polymorphism and vascular inflammatory disease.

Thrombosis and Behçet’s syndrome in non-endemic regions

Behçet’s syndrome (BS) is a rare, heterogeneous multisystem inflammatory condition, characterized by recurrent orogenital ulcers and uveitis. Vascular involvement affects 10–30% of patients, is most frequently observed in young males and usually presents as superficial or deep venous thrombosis (DVT) [1]. The spectrum of thrombosis also includes vena cava occlusion (inferior or superior), Budd–Chiari syndrome and dural sinus thrombi. Arterial aneurysms may occur and confer a serious bleeding risk, particularly when occurring in pulmonary arteries. Pulmonary artery aneurysms may also occur with DVT in isolation (Hughes–Stovin syndrome), although this is probably a forme fruste of BS [2]. In any event, the perceived increased risk of bleeding from aneurysms in association with venous thrombosis leads to a significant management conundrum. A number of studies have looked systematically for an intrinsic disorder of coagulation in BS, but no clinically useful correlates with thrombosis have been identified [3]. As the likely cause of thrombosis is venous inflammation, an immunosuppressive approach to management is therefore reasonable, although the evidence indicating specific treatments is very limited. Perhaps the best support comes from the well-cited 2-year randomized placebo-controlled double-blind trial of AZA in BS, in which analysis of the extra-ocular outcomes showed that the number of patients who developed DVT was fewer in the AZA arm [4]. What then of anticoagulation? The European League Against Rheumatism (EULAR) has recently published recommendations for the management of thrombosis in BS in which the use of corticosteroids and immunosuppressants (such as AZA, CSA or cyclophosphamide) is fully endorsed [5]. These guidelines discourage the use of anticoagulants and anti-platelet agents, both on account of the increased risk of bleeding and because protection from pulmonary embolism is seen as a low priority. Thus, pulmonary embolism in BS is thought by many to be unusual, perhaps because clots become tethered to the inflamed vessel wall. Some support for the EULAR guidelines on thrombosis in BS comes from a recent small study which compared the use of immune suppression and anticoagulation [6]. Thirty-seven patients with BS and venous thrombosis were retrospectively divided into three groups: immunosuppression alone (16 patients), combination immunosuppression and anticoagulation (17 patients) and anticoagulation alone (4 patients). Re-thrombosis was the study efficacy end-point. Three of four patients who were treated using warfarin without immunosuppression showed recurrence or progression of venous thrombosis, suggesting that anticoagulation therapy alone is not effective in treating DVT. As there were no significant differences in coagulation profile or the thrombosis recurrence rate in the immunosuppressant group and the combination therapy group, the authors suggested that anticoagulation in addition to immunosuppression may be unnecessary. We have recently reviewed vascular complications in patients with BS attending our tertiary referral centre practice between 1994 and 2009, and the results highlight the difficulties presented by this group of patients and call for a tempered view on the contraindication to anticoagulation in regions such as the UK where BS is not endemic. Of 657 patients reviewed, 62 (9%) had a history of thrombosis, consistent with other series. Among these 62 patients, thrombosis was the event that eventually led to the diagnosis of BS in 39, and 17 were considered to have had a pulmonary embolus. Importantly, the large majority [55 (89%) patients] were treated with warfarin at the time of thrombosis and before referral to our centre. Warfarin was discontinued because of complications in only two patients, one due to haemoptysis secondary to pulmonary aneurysms and the other due to an upper gastrointestinal bleed. Given (i) the rarity of BS in the UK; (ii) the lack of familiarity of most acute physicians with the condition; (iii) the frequent ambiguity of the diagnosis of BS in the UK; (iv) the chances of thrombosis in an undiagnosed patient being due to more common causes; and (v) the risk of pulmonary embolism in BS patients being as great, or greater, than that of bleeding, these patients were often, if not usually, appropriately managed. While accepting that the mainstay of treating thrombosis in BS is immunosuppression, the practical problems are often how best to limit the risk of anticoagulation and for how long to anticoagulate. Unquestionably, anticoagulation should be conducted in BS with extreme caution. Modern imaging provides an increasing armamentarium for screening the vasculature for aneurysms, and both CT and magnetic resonance angiography (MRA) are suitable. However, MRA is preferable if available, given the possible need for serial imaging and the need for minimizing radiation exposure. We