Molecular Analysis of Skin-Homing Receptors on Natural Killer Cells Using a Model of Allergic Contact Dermatitis
PI: Charles J. Dimitroff, Ph.D.
Introduction and Specific Aim
Migration of effector leukocytes into inflamed skin is a process, in part, controlled by adhesive interactions between leukocyte E-selectin ligands and dermal endothelial E-selectin. Unlike other microvascular endothelial cells, dermal microvascular endothelial cells constitutively express E-selectin (1-3). Expression of E-selectin is critical for dermal recruitment of leukocytes associated with damaged skin (4), chronic inflammation (5-9) and autoimmunity (10) and of pathologic leukocytes in cutaneous leukemias (11-14). E-selectin-binding determinants on leukocytes are comprised of α2,3 sialylated, α1,3-fucosylated oligosaccharides displayed on membrane glycoprotein(s) and are critical for dermal leukocytotropism. The functionality of these glycoconjugates signifies their designation as skin-homing receptors. Deletion of leukocyte α1,3 fucosyltransferases, which synthesize E-selectin-binding determinants, results in defective cutaneous inflammatory responses, underscoring the importance of selectin ligands in skin disorders, such as allergic contact hypersensitivity (CHS), atopic dermatitis, psoriasis, and cutaneous lymphomas (15). The cell-specific expression of dermal E-selectin and leukocyte E-selectin ligand is, therefore, an important feature in developing therapeutic strategies to selectively control the recruitment of lymphocytes associated with cutaneous inflammation and cancer. Indeed, studies testing the in vivo efficacy of a novel inhibitor of selectin ligand biosynthesis, 4-F-GlcNAc, indicates that inhibition of E-selectin ligand(s) on effector leukocytes in allergic CHS markedly reduces the inflammatory response (16, 17).
The most notable membrane selectin glycoprotein ligand on leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). In fact, PSGL-1 bearing E-selectin-binding determinants has been viewed as the skin-homing receptor on leukocytes due to its conspicuous expression on a variety of hematopoietic cell lines, in vitro-expanded T cells and on ex vivo T cells. Contrary to the contributing role of PSGL-1 as the principal E-selectin ligand on skin-homing leukocytes, recent studies show that PSGL-1 is dispensable for a fulminant allergic CHS response and that the major E-selectin ligand on skin-homing leukocytes is still unknown (20, 21).
Interestingly, von Andrian et al. and others show that natural killer (NK) cells can serve as important cellular mediators of allergic CHS responses (22, 23). They show that NK cells mediate hapten-specific CHS in Rag2-/- or SCID mice that are deficient in T and B cells (22, 23). This surprising function of NK cells in adaptive immune responses has stimulated research to dissect the molecular mediators of NK cell recruitment to inflamed skin, particularly in the pathologic context of cutaneous inflammation. Since dermal endothelial E- and P- selectin are critical for the trafficking of effector leukocytes to skin, we hypothesize that NK cell E and P-selectin ligands confer the skin-homing feature of NK cells.
The overall objective of this pilot and feasible project is to identify and characterize specific membrane scaffold(s) displaying E-selectin-binding determinants on freshly-isolated effector NK cells. Elucidating the E-selectin ligand repertoire on NK cells will undoubtedly improve our understanding of the pathogenesis of leukocyte-mediated dermatoses.
Specific Aim: To determine the protein identity of NK cell E-selectin ligands.
The role of Monocyte Chemoattractant Protein-2 and CCR8 in a mouse model of atopic dermatitis
PI: Sabina A. Islam, MD
Introduction and Specific Aim
Atopic dermatitis is a chronic inflammatory skin disorder typified by pruritic inflamed skin and is one of the most common inflammatory skin conditions in the world(1, 2). Dermatitis is characterized by aninflammatory leukocytic infiltrate in the dermis, epidermis and perivascular regions. The cells that infiltrate acute dermatitis lesions include Th2 cells, eosinophils, and mast cells that produce the Th2 cytokines IL-4, IL-5, IL-10, and IL-13, while Th1 cytokines IL-12 and IFNγare highly expressed in the chronic phase of the disease. The skin of the majority of atopic dermatitis patients is colonized with superantigen-producing strains of Staphylococcus aureus, thought to play a role in the pathogenesis of the disease(1, 3). Many chemokines have been detected at elevated levels in inflamed skin and sera of patients including TARC (CCL17), CTACK (CCL27) and TCA-3(CCL1), which respectively are ligands for CCR4, CCR10 and CCR8(2, 4, 5). Chemokines and their receptors provide signals for the entry of leukocytes into the skin, and thereby regulate leukocyte mediated skin inflammation. We are proposing to study the role of the T cell chemokine receptor CCR8 in regulating T cellaccumulation in the skin and the pathogenesis of allergic skin inflammation in a murine model of atopic dermatitis.
We have determined that mouse Monocyte Chemoattractant Protein-2 (MCP-2) is a novel second ligand for the mouse CCR8 receptor. In addition to Th2 cells and Tregulatory cells, CCR8 expression has been reported on memory CD8 and Th17 cells in humans(6-11). CCR8-gene-deficient mice are likely to have impaired trafficking of any or all of these T cell subsets. We have made novel observations that mouse MCP-2 mediates mouse Th17, Th2 CD4 and memory CD8 cell migration and calcium flux in vitro at physiologically relevant concentrations. Using gene knockout cells, antibody inhibition studies and transfectants, we have determined that mouse MCP-2 (CCL8) is a second ligand for mouse CCR8. The receptor for mouse MCP-2 has not been fully characterized previously. The Monocyte Chemoattractant Protein familyof chemokines are not orthologousacross mammalian species. Human MCP-2 has been characterized and is a functional ligand for the human chemokine receptors CCR1, CCR2, CCR3 and CCR5(12-14). However mouse MCP-2 does not activate these mouse receptors at physiologically relevant concentrations; it has been reported that very high concentrations of mouse MCP-2 are required to induce Ca flux of murine CCR2 transfectants. We also find that MCP-2 is very highly expressed in the skin of healthy mice at both mRNA and protein levels. mRNA expression of mouse MCP-2 in healthy skin is second only to that of CTACK. Recently, investigators have also reported that MCP-2 is dramatically induced in the skin in a murine model of atopic dermatitis(3). Based on the above experimental findings we induced atopic dermatitis in WT C57Bl/6 mice and CCR8-gene-deficient mice by epicutaneous sensitization. We have now made the novel observation that though allergen sensitization induces the atopicdermatitis disease phenotype in WT C57Bl/6 mice, CCR8-gene-deficient mice are protected. This leads us to hypothesize that MCP-2 regulates CCR8 mediated T cell trafficking to skin and that the newly described MCP-2-CCR8 pathway is critical for disease induction in a murine model of atopic dermatitis. We therefore wish to pursue the following:
Specific Aim: To determine how CCR8 and its novel ligand MCP-2 regulate T cell subset accumulation ininflamed allergic skin and contribute to disease pathogenesis in a mouse model of atopic dermatitis.
Using the epicutaneous sensitization model of murine atopic dermatitis, we find that CCR8-deficient mice have a striking reduction in epidermal and dermal thickening, and reduced cellular infiltration into skin on hematoxylin and eosin staining compared to wild-type C57Bl/6 mice. In this aim we intend to use this murine model of atopic dermatitis to:
- Fully characterize and compare CCR8-deficient and wild-type C57Bl/6 mice that undergo epicutaneous sensitization by comprehensive immunohistochemical and immunological parameters to define differences in pathogenic leukocyte subset recruitment to inflamed skin and differences in T celleffector functions.
- Perform a kinetic analysis of Th1, Th2, Th17, T regulatory CD4 cell and IL-13 producing CD8 memory/effector T cell induction in draining lymph nodes and skin of CCR8-deficient and wild-type mice during epicutaneous allergen sensitization.
- Perform adoptive transfer studies to compare the trafficking of transferred wild-type-OT-II TCR transgenic T cells and CCR8-deficient-OT-II TCR transgenic T cells that recognize the OVA antigen in congenic wild-type mice that will undergo epicutaneous OVA sensitization for induction of allergicskin inflammation.
- Characterize the phenotype of this model in MCP-2-deficient mice to definitively establish the importance of the mouse MCP-2-CCR8 pathway in T cell trafficking to allergic skin in vivo.
- Determine whether CCR2-gene-deficient mice are protected from allergic skin inflammation in themurine model of atopic dermatitis to discern whether mouse MCP-2-CCR2 signaling is functionally relevant in vivo.
Role of IL-1 in cutaneous immune response to vaccinia virus
PI: Tian Tian, MD Ph.D.
Introduction and Specific Aims
Smallpox is a highly contagious and often fatal disease caused by infection with variola virus. Smallpox outbreaks have occurred throughout the world over thousands of years. There is no specific treatment of this disease, only prevention via vaccination. As the result of an aggressive global vaccination program, smallpox was eradicated from the world as a naturally occurring disease in 1977. Consequently, routine vaccination against smallpox was discontinued in the
The anthrax attacks in the
Vaccinia virus (VV), a virus related to variola virus, is used as the primary smallpox vaccine. Although most immunizations are delivered by either subcutaneous or intramuscular injection, effective smallpox vaccination currently requires inoculation of live vaccinia virus into the skin via scarification, leading to an epidermal pox reaction. Vaccinia scarification in a normal host results in specific and long-lasting immunity; however, in atopic and immunodeficient people, including the very old and the very young, viral replication can outstrip control, leading to devastating morbidity and mortality (Bray M, 2003). These adverse events may relate to a failure of the host immune system to control vaccinia virus replication and dissemination. We know from previous work in cutaneous immunobiology that the balance and interaction of innate and acquired immune defense mechanisms at the cutaneous interface are crucial elements in determining the speed and character of the immune response to inoculation (Kupper TS, 2004, Howell MD, 2006). Thus, manipulation of the microenvironment of inoculated skin, such as modulating local chemokine or cytokine expression, could alter the immune response to viral challenge.
IL-1 is an important regulator of cutaneous inflammatory responses and contributes to the host immune defense against infection. Interestingly, IL-1-related proteins are encoded by poxvirus genomes and can modulate the host immune response to viral infection (Alcami A, 2003). For example, the VV gene B15R encodes soluble IL-1 receptor, which binds IL-1β. Deletion of B15R from VV accelerates the appearance of symptoms of illness and mortality in mice infected intranasally. These results support the following hypotheses regarding the role of IL-1 in cutaneous immunity against VV: 1) Manipulation of IL-1 in vivo can control cutaneous immunity to VV, 2) IL-1 acts to modify cutaneous immunity to VV via dendritic cells (DC), and 3) exogenous IL-1 can be used as adjuvant to enhance cutaneous immunity to VV.
To test these hypotheses, we propose the following specific aims:
Aim1. To determine whether manipulation of IL-1 in vivo alters cutaneous immunity to VV using Tg mice that overexpress IL-1α, IL-1 Receptor 1 (IL-1R1) or IL-1 Receptor2 (IL-1R2) in the epidermis
Aim2. To determine whether IL-1 regulates cutaneous immunity to VV though DC using CD11c/DTR mice
Aim3. To determine whether exogenous IL-1 can be used as an adjuvant to enhance cutaneous immunity to VV.
Deriving cells of the keratinocyte lineage from hES cell lines: toward proof-of-principal for therapeutic use of hES-derived somatic cells
PI: James Rheinwald, MD Ph.D
Since their first cultivation and initial characterization (for example, see Thomson et al., 1998; Shamblott et al., 2001; Cowan et al., 2004), human embryonic stem (hES) cell lines have received much interest for elucidating mechanisms of somatic lineage formation during development and for their potential to produce functional cells for cell replacement therapy (reviewed by Odorico et al., 2001). Space limitations here do not permit a summary of the many reports of the ability of hES lines to produce embryoid bodies or teratomas containing clusters of cells expressing markers of endoderm-, ectoderm-, and mesoderm-derived lineages. In contrast, however, there are very few reports of isolation of even modestly-proliferative, and partially-purified populations derived from hES cells that display features of a specific somatic cell type (Levenberg et al., 2002; Xu et al., 2002; Green et al., 2003; Lavon et al., 2004; Wang et al., 2005). The ideal strategy, envisioned to avoid problems of allograft rejection, is “therapeutic cloning”---the generation of custom hES lines by somatic cell nuclear transfer into unfertilized oocytes, from which hES lines will be produced and, in turn, give rise to a desired somatic cell type for transplantation into the individual who had been the source of the somatic cell nucleus. Such hES lines could be genetically engineered, if necessary, to repair a genetic defect of the intended recipient before production of differentiated cells and transplantation (Zwaka and Thomson, 2003; Urbach et al., 2004). Although the inner cell mass cells of different blastocyst-stage embryos are likely to be very similar in their properties, serially passaged hES lines may vary in their differentiation potential and may also acquire genetic and epigenetic abnormalities as the result of selection for more rapidly dividing variants during serial passage (Inunza et al., 2004). These changes may interfere with the ability of some lines to form certain lineages or may otherwise preclude use in cell replacement therapy. Before hES-derived cells can be used routinely in the clinic, it is essential to assess the cell lineage-forming potential of existing and newly generated hES lines, to isolate pure populations of the desired somatic cell type, and to demonstrate proof-of-principal for their function in vivo in preclinical models.
My lab and others have optimized culture conditions for clonal growth and expansion for ~40-70 population doublings of normal human keratinocytes from the many types of stratified squamous epithelial tissue (e.g., epidermal, corneal, esophageal, ectocervical, etc.) (Rheinwald and Green, 1975; Banks-Schlegel and Green, 1981; Wu et al., 1982; Allen-Hoffmann and Rheinwald, 1984; Dickson et al., 2000). Markers have been identified for the specific pattern of terminal differentiation to which the keratinocyte “subtypes” that form each of these epithelia become directed during development (Wu et al., 1982; O’Guinn et al., 2001; Lindberg and Rheinwald, 1990; Lindberg et al., 1993). We have identified a p16INK4A-enforced mechanism that growth-arrests keratinocytes under certain pathologic situations in vivo and that operates independently from telomere erosion to limit expansion potential of primary keratinocytes in culture (Dickson et al., 2000; Rheinwald et al., 2002; Natarajan et al., 2005). We have recently compiled an extensive microarray database of the gene expression profiles of normal human keratinocytes in culture (Guo, Rheinwald et al., in preparation). Organotypic culture and nude mouse xenograft systems have been developed that permit cultured human keratinocytes to form a stratified epithelium identical to that in vivo (Barrandon et al.,1988; Lindberg and Rheinwald, 1990; Lindberg et al., 1993; Garlick and Taichman, 1994; Dickson et al., 2000), thereby providing experimental systems for assessing histogenic potential of this cell type. Importantly, normal epidermal and corneal keratinocytes remain able after considerable expansion in culture to reestablish permanently renewing, functional epithelia as autologous transplants (Gallico et al., 1984; Compton et al., 1989; Pelligrini et al., 1997).
The production from hES cells of certain somatic cell types, such as pancreatic islet cells and neuronal progenitor cells, may be of more pressing clinical need than the formation of keratinocytes. However, the depth and breadth of knowledge of keratinocyte biology, the impressive proliferative potential of this cell type in culture, and its ability to regenerate a permanent, normally functioning tissue in autologous transplants provides a strong experimental foundation and rationale for pursuing this cell type to obtain proof-of-principal for hES-derived cell therapy. The project outlined here will optimize conditions for generating keratinocytes from hES cells, permit a more thorough assessment of differentiation potential for various hES cell lines, and demonstrate proof-of-principal for generating a functional somatic cell type from hES cell lines. Charbel Bouez, the postdoctoral fellow in my lab responsible for this project, is very experienced in keratinocyte culture, retroviral and lentiviral transduction, and organotypic culture and also has taken the one week intensive course in hES cell culture given by Thorsten Schlager of the stem cell core of Children‘s Hospital and the HSCI.
