Endothelial Cell: T Cell Interactions

Altieri, Askenase, Bender, Bothwell, Braverman, *Kluger, Madri, *McNiff, Pober, *Rothstein, *Schechner, Tellides

Dario Altieri (Pathology) has two principal research interests: 1) leukocyte and endothelial cell interactions with protein cascades in blood coagulation and 2) structure-function characterization of a novel IAP inhibitor of apoptosis, designated survivin. A major focus of the first interest has been the detailed study of effector cell protease receptor-1 (EPR-1), the major receptor for factor Xa on endothelial cells. His group has recently demonstrated that in vivo injection of factor Xa (or a synthetic peptide mimicking the EPR-1 binding domain on Xa) resulted in an acute inflammatory response, consistent with the possibility that Xa-EPR-1 interactions may amplify both coagulation and inflammatory cascades, and thereby contributing to the pathogenesis of tissue injury in vivo. Very recent studies have shown that the signaling pathway(s) initiated by factor Xa binding to ERP-1 on endothelial cells lead to strikingly increased release of nitric oxide, and that such release in vivo is necessary both for the inflammatory response described above as well as for the hypotension (vasorelaxation) that accompanies in vivo binding of factor Xa to endothelial cell EPR-1. Furthermore, they have also shown that targeting EPR-1 with antisense oligonucleotide or with a monoclonal antibody inhibited CD3/T cell receptor-dependent lymphocyte activation, indicating a new role for protease receptors in the regulation of the immune response, and identifying a potential target for therapeutic immunosuppression in humans.

The second major research focus in Dr. Altieri's laboratory is on survivin, an IAP inhibitor of apoptosis which was very recently identified and cloned in his laboratory. Survivin is undetectable in normal terminally differentiated tissues and becomes abundantly re-expressed in a variety of human cancers, in vivo. In addition to beginning an examination of survivin expression in melanoma (see also Epidermal Biology group, Melanocytes/Melanoma subcluster), Dr. Altieri has also very recently become interested in investigating the expression of survivin in endothelial cells of skin lesions containing either normal or malignant proliferation of endothelial cells (e.g., angioma, angiosarcoma, Kaposi's sarcoma) or vasocentered inflammation (e.g., leukocytoclastic or lymphocytic vasculitis). He also has been discussing with Dr. Kluger a future collaboration to investigate the consequences of stable overexpression of survivin in cultured HDMECs in order to test whether its anti-apoptotic effects confer resistance to such cells against killing by cytotoxic T cells, activated neutrophils, and a variety of other potentially toxic insults.

Dr. Philip Askenase received YSDRC P/F study funding during the 02-3 years of this Center; the goal of this study, carried out in collaboration with Dr. Jordan Pober (Pathology) was to develop the human:scid mouse skin/PBL chimera model for the study of allograft reactions and, ultimately, for the study of delayed hypersensitivity reactions (including contact dermatitis) in chimeric mice bearing skin grafts syngeneic with the human PBL. These ongoing studies have resulted in three publications (see also Dr. Bothwell, immediately below), and have been pivotal in the subsequent expansion in this revised application of this powerful model system into a YSDRC Core Facility.

Dr. Jeffrey Bender's research interests include the study of dermal microvascular endothelium and its susceptibility to inflammatory changes. His group has characterized cytokine-induced adhesion molecule expression in micro- and large vessel endothelial cells. They have also defined unique aspects of dermal microvascular endothelial cell junctions which are affected by inflammatory mediators and result in alteration of barrier properties. As an investigator in the Molecular Cardiobiology Unit headed by Dr. Jordan Pober, Dr. Bender interacts on a daily basis with investigators in Dr. Pober's group. He was also a co-investigator on Dr. Leonard Milstone's (Dermatology) P/F project to investigate epican (CD44) expression on keratinocytes and its role in cell:cell adhesion. His laboratory has recently demonstrated the abundance of caveolae in human dermal microvascular endothelial cells, and the role of these signaling organelles in cytokine--mediated endothelial signal transduction. Working with Drs. Pober and Schechner, Dr. Bender has begun utilizing the reconstituted SCID mouse model, evaluating role of human natural killer lymphocytes in vascular injury and rejection of both human and pig skin grafts; these studies utilize the services of the proposed YSDRC SCID Mouse:Skin Xenograft Core.

Dr. Alfred Bothwell has been actively collaborating with Dr. Jordan Pober in studies which have shown that human T cells show a strong xenogeneic response to porcine endothelial cells, and that the CD2 and CD28 pathways are major factors in this response. The pB7.2 antigen contributes to this response and the cDNA has been cloned and is being studied in functional assays. A very recent productive collaborative study involving Drs. Bothwell, Askenase, McNiff and Pober investigated T cell responses to porcine skin grafts on SCID mice reconstituted with human peripheral blood cells, and demonstrated that pig, but not human, IFN-g initiated rejection. Using this model, they are hoping to develop additional analytic systems to evaluate this interaction, such as the growth of porcine endothelial cells in 3-dimensional Matrigel cultures which will then be implanted into hu-SCID mice. One goal of this system is to evaluate the potential of porcine cells as a source of donor cells for xenografts.

Dr. Irwin Braverman has had long-standing interests in cutaneous microvasculature, and his studies utilizing laser doppler-computer topographic mapping of the cutaneous microcirculation permitted reliable identification of four major areas in the upper plexus: sites with both arterioles and venules, sites of arteriolar predominance, sites of venular predominance, and relatively avascular sites. His efforts have involved quantitating the response of vasomotion at each site to a variety of physiologic stimuli such as heat, cold, reactive hyperemia and vasoactive drugs. Responses in normal subjects were compared to those in patients with microvascular diseases such as scleroderma, diabetes mellitus and venous insufficiency. More recently, he was involved in a productive collaboration with Dr. Jordan Pober and Dr. Peter Petzelbauer examining skin microvascular alterations in psoriasis, and another productive collaboration with Dr. William Sessa (Pharmacology and Boyer Center Molecular Cardiobiology Section)demonstrated for the first time that efficient synthesis of nitric oxide required endothelial nitric oxide synthase to be Golgi-associated; both of these studies utilized the EM facilities offered until 1997 in the YSDRC's TAD/BASF Core

Dr. Jordan Pober (Pathology, Immunobiology and Dermatology) heads the Molecular Cardiobiology section in the Boyer Center for Molecular Medicine, and is an internationally recognized immunopathologist with particular interests/expertise in endothelial cell:immune system interactions. His laboratory studies the roles of vascular endothelial cells in immunity and inflammation, and have concentrated on skin microvessels for many of their studies. Their studies have focused upon how individual and combinations of cytokines alter endothelial surface proteins which interact with T cells, particularly MHC molecules and lymphocyte adhesion proteins. They are also examining the second messenger pathways utilized by cytokines to alter surface protein expression as well as the nuclear events mediated by cytokines that lead to altered rates of gene transcription. One of Dr. Pober's initial YSDRC interactions involved the postdoctoral training of Dr. Peter Petzelbauer, who subsequently returned to the Dermatology Department at the University of Vienna as a junior faculty member. Dr. Petzelbauer's studies included examining the differences among endothelial cells located in different segments of a single microvascular bed. work, as well as a collaborative study with Dr Braverman analyzing endothelial cell adhesion molecule expression in psoriatic skin. His next dermatology postdoctoral fellow, Dr. Jeffrey Schechner, M.D., (now an Assistant Professor of Dermatology) focused his attentions primarily on 1) studying the interactions between endothelial cells and malignant T cells (during the course of which he developed substantial technical expertise in the preparation of SCID mouse :skin xenografts) and 2) investigating strategies to facilitate tube formation by cultured endothelial cells and conditions for successful anastomosis with host vessels when implanted into SCID mice. Dr. Martin Kluger, Ph.D, who has very recently (7/1/98) joined the faculty in the Department of Dermatology as an Associate Research Scientist, also spent three productive years as a postdoctoral fellow in Dr. Pober's laboratory, examining mechanisms by which cytokine activation of dermal microvascular endothelial cells (HDMECs) results in their sustained cell surface expression of E-selectin. During the course of these studies, Dr. Kluger developed substantial proficiency in culturing both HDMECs and HUVECs; these skills made him the obvious choice as Co-Director of the proposed Cell Culture Core's endothelial cell subcore.

Dr. Pober's interactions with Dr. Askenase in developing the SCID mouse model using human skin xenografts that retain human skin microvessels in order to experimentally manipulate the functions of human endothelial cells in normal and transplantation immunity in vivo have been mentioned above; this YSDRC-funded P/F study facilitated the development of this model system to a point that this facility has been established as a YSDRC Core Facility which has already been utilized by a significant number of YSDRC investigators with broad-ranging interests. Finally, his ongoing collaborative association with Dr. Bothwell and Dr. Madison in the study of human anti-pig xenograft rejection both in vitro and in vivo (using the SCID mouse model) has also been described above.

Joseph Madri's research group (Pathology) has for many years studied vascular cell homotypic and heterotypic interactions and vascular cell interactions with the surrounding matrix, soluble factors and circulating blood cells (leukocytes, monocytes and Iymphocytes). While such interactions, at least in part, direct the vascular cells' responses, our understanding of these complex interactions and the mechanisms involved in information transduction from the extracellular environment to the cell nucleus is far from complete. Dr. Madri and his co-workers are currently investigating the roles of selected homotypic and heterotypic cell adhesion molecules (PECAM 1), integrins a1 to 6, b1 as well as heterodimers of the b2 and b3 integrin classes, junction associated molecules and extracellular matrix components and their alternative splicing patterns in modulating vascular cell and T cell behavior attachment, spreading, proliferation, migration, interactions with mononuclear (T) cells and angiogenesis. Current projects in his laboratory: 1 ) Elucidation of the roles of the cytoplasmic domains of Platelet Endothelial Cell Adhesion Molecule 1 (PECAM 1) in modulating endothelial and T cell behavior, vascular development and dermal wound healing as well as the signaling mechanisms involved. Matrix dependent, transient differential tyrosine and serine phosphorylation has been documented in an ITAM domain of PECAM 1 as well as associations with specific proteins including b-catenin and SHP 2. 2) Identification and elucidation of the signal transduction pathways activated following T cell a4b1 integrin endothelial cell VCAM 1 binding prior to, during and following T cell transmigration into tissues. Specific proteinase induction in T cells (matrix metalloproteinase 2 [MMP 2 ] and membrane type matrix metalloproteinase 1 [MT1 MMP]) has been found associated with engagement of this receptor pair and the signaling pathways involved in this process are being investigated using antigen specific T cell clones and COS cells transfected with (a4 integrin and MMP 2 promoter luciferase constructs. 3) Elucidation of the roles of PDGF and TGFD isoforms as morphogens and mitogens in the processes of vasculogenesis and dermal angiogenesis in wound healing and their receptor isoform ratios in these processes. These studies have involved productive collaboration with several YSDRC investigators, including Charles Janeway, Michael Centrella, and Thomas McCarthy.

Dr. Jennifer Madison McNiff , Director of the Section of Dermatopathology and of the YSDRC Tissue Acquisition and Analysis Core, joined the Department of Dermatology less than five years ago, and despite significant service obligations has promptly established a wide range of productive collaborative associations that utilize her outstanding dermatopathology skills. Current interactions include: 1) Studies with Drs. Askenase, Bothwell, Pober and Schechner utilizing the SCID mouse model to investigate pig:human xenograft reactivity in vivo as well as to determine the roles of various endothelial cell adhesion molecules in skin allograft rejection in the SCID mouse: human skin/human PBL model ; 2) Collaboration with Dr. Jeffrey Schechner in his immunohistochemical analysis of adhesion molecule expression both in biopsies from CTCL patients and in SCID mice bearing human skin and reconstituted with PBLs from leukemic CTCL patients; 3) Collaboration with Alan Garen (Molecular Biochemistry & Biophysics) in the development of melanoma vaccines by screening of human melanoma tissue with potential melanoma-specific antibodies selected from monoclonal antibody libraries; 4) Studies with John Pawelek (Dermatology) involving analysis of melanoma-macrophage hybrid cell lines and immunophenotyping of melanomas for macrophage characteristics; 5) Collaboration with Dr. Henry Spinelli (Plastic Surgery) in studies of the role of rotational orientation on microvascular anastamoses; 6) Collaboration with Drs. Cappello (Pediatrics) and Bromberg (Hematology) in their studies (P/F # 17) of the role of tissue factor in the metastatic potential of malignant melanoma; 7) Collaboration with Drs. Joseph Craft (Rheumatology)and Mark Shlomchik (Laboratory Medicine) investigating the lupus-like skin lesions in a variety of strains of mice bred to be both genetically deficient in selected components of their immune system and autoimmune-prone (MRL/lpr). P/F project (#23).

Dr. David Rothstein (Nephrology) is currently studying the role of the CD45 tyrosine phosphatase in the induction of transplantation tolerance. He has shown that treatment with three doses of anti-CD45 mAb induces long-term tolerance in 50% of murine islet allograft recipients. Effective anti-CD45RB mAbs appear to function by altering CD45 isoform expression, upregulating the expression of low Mr CD45 isoforms that are expressed by CD4 cells secreting Th2-type cytokines and having distinct downregulatory activity in autoimmune models. Furthermore, anti-CD45 mAbs alter T cell activation :signal one" and may thereby induce anergy. Combining blockade of "signal one" with blockade of "signal 2" (with anti-CD40L) boosts long-term graft acceptance to 85%. He is now examining tolerance to both skin and islet allografts in animals treated with these potent immunotherapeutic agents, alone or in combination.

Based on promising results in murine allografts, he has recently initiated studies using the SCID mouse: human skin/allogeneic human PBL model to examinethe prevention of human skin (and islet cell) allograft rejection. Use of the SCID model will be critical in allowing him to identify the most effective mAbs against human CD45 and helping him determine which CD isoforms are best targeted. Such studies will obviously be necessary before preclinical studies in humans are envisioned. These studies will be performed in collaboration with Drs. Pober and Schechner in the YSDRC SCID Mouse:Skin Xenograft Core.

Dr. Jeffrey Schechner (Dermatology) is an Assistant Professor of Dermatology and has been identified in this revised application as the Co-Director of the SCID Mouse : Skin Xenograft Core, based on his extensive use of this model over the past four years and his consequent understanding of the technical subtleties necessary to successful engraftment of xenogeneic skin and (subsets) of lymphoid cells. His research has focused on the expression of adhesion molecules on lymphocytes, endothelial cells and epithelial cells, and what role they may play in the infiltration of the skin by lymphocytes in benign inflammatory dermatoses and CTCL. A collaborative study involving Drs. Pober, Heald, and Madison directed toward identifying endothelial cell and lymphocyte adhesion molecules that participate in infiltration of the skin by CTCL cells and making extensive use of the SCID mouse model, was submitted as a P/F project in this Center's competitive renewal application (see CTCL cluster above).

Finally, the major objective of research in the laboratory of Dr. George Tellides (Cardiothoracic Surgery) is to create in vivo models to study the immunobiology of human cellular immune responses to pig xenografts. The models consist of pig skin grafts or pig coronary artery grafts transplanted to SCID mice which are immunologically reconstituted with human peripheral blood mononuclear cells (huPBMC). This project has four specific aims: First, to complete the development of pig skin and coronary artery xenograft transplantation models in chimeric huPBMC-SCID/beige mice which were established in preliminary experiments done in collaboration with Drs. Jordan Pober and Jeffrey Schechner. Second, to characterize the endothelial and immune responses at various stages of rejection in this model. Skin graft rejection and coronary artery graft patency are evaluated macroscopically. Evidence for vascular injury and inflammatory cell infiltration is assessed by light microscopy. Porcine endothelial cell activation, the species and phenotype of infiltrating cells, and intra-graft cytokine expression is determined by immunohistology. (These analyses will involve utilization of the services of the Tissue Acquisition and Analysis Core.) Third, to assess the capacity of various human immune cell populations to mediate xenograft rejection. Specific cells are depleted in the PBMC inoculum using appropriate monoclonal antibodies conjugated to magnetic beads. The immunopathologic consequences following reconstitution with NK cells, T and B lymphocytes, and various T cell subsets are determined as described immediately above. Fourth, to evaluate the effects of immuno- suppressive agents on human cell-mediated injury of pig endothelium. Several pharmacologic agents or monoclonal antibodies will be administered and their efficacy determined.