ABSTRACT
Langerhans cells (LCs) are epidermal dendritic cells with incompletely understood origins that associate with hair follicles for unknown reasons. Here we show that in response to external stress, mouse hair follicles recruited Gr-1(hi) monocyte-derived precursors of LCs whose epidermal entry was dependent on the chemokine receptors CCR2 and CCR6, whereas the chemokine receptor CCR8 inhibited the recruitment of LCs. Distinct hair-follicle regions had differences in their expression of ligands for CCR2 and CCR6. The isthmus expressed the chemokine CCL2; the infundibulum expressed the chemokine CCL20; and keratinocytes in the bulge produced the chemokine CCL8, which is the ligand for CCR8. Thus, distinct hair-follicle keratinocyte subpopulations promoted or inhibited repopulation with LCs via differences in chemokine production, a feature also noted in humans. Pre-LCs failed to enter hairless skin in mice or humans, which establishes hair follicles as portals for LCs.
Subject(s)
Chemokines/biosynthesis , Hair Follicle/immunology , Langerhans Cells/physiology , Stress, Physiological , Alopecia , Animals , Cell Movement , Chemokine CCL20/biosynthesis , Chemokine CCL8/biosynthesis , Chemokines/metabolism , Hair Follicle/metabolism , Humans , Keratinocytes/metabolism , Langerhans Cells/immunology , Mice , Mice, Hairless , Receptors, CCR2/metabolism , Receptors, CCR6/metabolism , Receptors, CCR8/metabolism , Skin/immunologyABSTRACT
Mechanisms responsible for protective immunity against epicutaneous Candida infections are incompletely characterized. In this issue of Immunity, Kashem et al. demonstrate that different Candida life forms engage selected skin dendritic cell subsets in distinct compartments, resulting in qualitatively different immune responses.
Subject(s)
Candidiasis, Chronic Mucocutaneous/immunology , Cell Differentiation/immunology , Dendritic Cells/immunology , Th17 Cells/cytology , Th17 Cells/immunology , AnimalsABSTRACT
Thymic stromal lymphopoietin (TSLP) is a type I cytokine that plays a central role in induction of allergic inflammatory responses. Its principal targets have been reported to be dendritic cells and/or CD4 T cells; epithelial cells are a principal source. We report in this study the development of a reporter mouse (TSLP-ZsG) in which a ZsGreen (ZsG)-encoding construct has been inserted by recombineering into a bacterial artificial chromosome immediately at the translation initiating ATG of TSLP. The expression of ZsG by mice transgenic for the recombinant BAC appears to be a faithful surrogate for TSLP expression, particularly in keratinocytes and medullary thymic epithelial cells. Limited ZsG and TSLP mRNA was observed in bone marrow-derived mast cells, basophils, and dendritic cells. Using the TSLP-ZsG reporter mouse, we show that TNF-α and IL-4/IL-13 are potent inducers of TSLP expression by keratinocytes and that local activation of Th2 and Th1 cells induces keratinocyte TSLP expression. We suggest that the capacity of TSLP to both induce Th2 differentiation and to be induced by activated Th2 cells raises the possibility that TSLP may be involved in a positive feedback loop to enhance allergic inflammatory conditions.
Subject(s)
Cytokines/genetics , Gene Expression , Animals , Basophils/metabolism , Cholecalciferol/pharmacology , Cytokines/metabolism , Dendritic Cells/metabolism , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Gene Order , Genes, Reporter , Genetic Vectors/genetics , Interleukin-13/pharmacology , Interleukin-4/pharmacology , Keratinocytes/drug effects , Keratinocytes/metabolism , Lymphocyte Activation/immunology , Mast Cells/metabolism , Mice , Mice, Transgenic , Recombinant Fusion Proteins/genetics , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/immunology , Th2 Cells/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Thymic Stromal LymphopoietinABSTRACT
Psoriasis is a chronic inflammatory skin disease characterized by abnormal keratinocyte proliferation and differentiation and by an influx of inflammatory cells. The mechanisms underlying psoriasis in humans and in mouse models are poorly understood, although evidence strongly points to crucial contributions of IL-17 cytokines, which signal via the obligatory adaptor CIKS/Act1. Here we identify critical roles of CIKS/Act1-mediated signaling in imiquimod-induced psoriatic inflammation, a mouse model that shares features with the human disease. We found that IL-17 cytokines/CIKS-mediated signaling into keratinocytes is essential for neutrophilic microabscess formation and contributes to hyperproliferation and markedly attenuated differentiation of keratinocytes, at least in part via direct effects. In contrast, IL-17 cytokines/CIKS-mediated signaling into nonkeratinocytes, particularly into dermal fibroblasts, promotes cellular infiltration and, importantly, leads to enhanced the accumulation of IL-17-producing γδT cells in skin, comprising a positive feed-forward mechanism. Thus, CIKS-mediated signaling is central in the development of both dermal and epidermal hallmarks of psoriasis, inducing distinct pathologies via target cell-specific effects. CIKS-mediated signaling represents a potential therapeutic target in psoriasis.
Subject(s)
Interleukin-17/immunology , Psoriasis/immunology , Aminoquinolines/administration & dosage , Animals , Cell Differentiation/immunology , Cell Proliferation , Disease Models, Animal , Epidermis/drug effects , Epidermis/pathology , Imiquimod , Mice , Mice, Inbred C57BL , Mice, Knockout , Psoriasis/chemically induced , Real-Time Polymerase Chain ReactionABSTRACT
The glycoprotein milk fat globule-epidermal growth factor factor 8 (MFG-E8) is expressed in several tissues and mediates diverse homeostatic functions. However, whether it plays a role in bone homeostasis has not been established. In this study, we show for the first time, to our knowledge, that osteoclasts express and are regulated by MFG-E8. Bone marrow-derived osteoclast precursors from MFG-E8-deficient (Mfge8(-/-)) mice underwent increased receptor activator of NF-κB ligand-induced osteoclastogenesis, leading to enhanced resorption pit formation compared with wild-type controls. Consistently, exogenously added MFG-E8 inhibited receptor activator of NF-κB ligand-induced osteoclastogenesis from mouse or human osteoclast precursors. Upon induction of experimental periodontitis, an oral inflammatory disease characterized by loss of bone support of the dentition, Mfge8(-/-) mice exhibited higher numbers of osteoclasts and more bone loss than did wild-type controls. Accordingly, local microinjection of anti-MFG-E8 mAb exacerbated periodontal bone loss in wild-type mice. Conversely, microinjection of MFG-E8 inhibited bone loss in experimental mouse periodontitis. In comparison with wild-type controls, Mfge8(-/-) mice also experienced >60% more naturally occurring chronic periodontal bone loss. In conclusion, MFG-E8 is a novel homeostatic regulator of osteoclasts that could be exploited therapeutically to treat periodontitis and perhaps other immunological disorders associated with inflammatory bone loss.
Subject(s)
Antigens, Surface/physiology , Bone Resorption/immunology , Homeostasis/immunology , Inflammation Mediators/physiology , Osteoclasts/immunology , Osteoclasts/pathology , Animals , Antigens, Surface/biosynthesis , Antigens, Surface/genetics , Bone Resorption/genetics , Bone Resorption/pathology , Cell Line , Homeostasis/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Milk Proteins/biosynthesis , Milk Proteins/genetics , Periodontitis/genetics , Periodontitis/immunology , Periodontitis/pathology , RANK Ligand/administration & dosage , RANK Ligand/adverse effects , Stem Cells/immunology , Stem Cells/metabolism , Stem Cells/pathologyABSTRACT
Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200(+) CD45(-) BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200(+) CD45(-) BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200(+) CD45(-) cells, representing ~1,500-fold enrichment. CD200(-) CD45(-) BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC.
Subject(s)
Antigens, CD/metabolism , Carcinoma, Basal Cell/immunology , Carcinoma, Basal Cell/pathology , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Animals , Carcinoma, Basal Cell/metabolism , Cell Differentiation , Cell Proliferation , Humans , Keratins/metabolism , Kruppel-Like Factor 4 , Mice , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Receptors, G-Protein-Coupled/antagonists & inhibitors , Skin Neoplasms/metabolism , Smoothened Receptor , Transplantation, Heterologous , Tumor Stem Cell AssayABSTRACT
Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 µM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 µM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells.
Subject(s)
Antigens, CD/metabolism , Bacterial Proteins/metabolism , Glycoproteins/metabolism , Lectins, C-Type/metabolism , Mannose-Binding Lectins/metabolism , Mycobacterium leprae/metabolism , Superoxide Dismutase/metabolism , Blotting, Western , Cell Wall/metabolism , Humans , Protein Binding , Surface Plasmon ResonanceABSTRACT
Our research group recently demonstrated that pericytes are major sources of the secreted glycoprotein and integrin ligand lactadherin (MFG-E8) in B16 melanoma tumors, and that MFG-E8 promotes angiogenesis via enhanced PDGF-PDGFRß signaling mediated by integrin-growth factor receptor crosstalk. However, sources of MFG-E8 and its possible roles in skin physiology are not well characterized. The objective of this study was to characterize the involvement of MFG-E8 in skin wound healing. In the dermis of normal murine and human skin, accumulations of MFG-E8 were found around CD31(+) blood vessels, and MFG-E8 colocalized with PDGFRß(+), αSMA(+), and NG2(+) pericytes. MFG-E8 protein and mRNA levels were elevated in the dermis during full-thickness wound healing in mice. MFG-E8 was diffusely present in granulation tissue and was localized around blood vessels. Wound healing was delayed in MFG-E8 knockout mice, compared with the wild type, and myofibroblast and vessel numbers in wound areas were significantly reduced in knockout mice. Inhibition of MFG-E8 production with siRNA attenuated the formation of capillary-like structures in vitro. Expression of MFG-E8 in fibrous human granulation tissue with scant blood vessels was less than that in granulation tissue with many blood vessels. These findings suggest that MFG-E8 promotes cutaneous wound healing by enhancing angiogenesis.
Subject(s)
Antigens, Surface/physiology , Dermis/physiology , Neovascularization, Physiologic , Skin Physiological Phenomena , Wound Healing , Animals , Cell Line , Granulation Tissue/physiology , Human Umbilical Vein Endothelial Cells/cytology , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Milk Proteins , Myocytes, Smooth Muscle/cytology , Myofibroblasts/cytology , Pericytes/cytologyABSTRACT
After activation, Langerhans cells (LC), a distinct subpopulation of epidermis-resident dendritic cells, migrate from skin to lymph nodes where they regulate the magnitude and quality of immune responses initiated by epicutaneously applied antigens. Modulation of LC-keratinocyte adhesion is likely to be central to regulation of LC migration. LC express high levels of epithelial cell adhesion molecule (EpCAM; CD326), a cell-surface protein that is characteristic of some epithelia and many carcinomas and that has been implicated in intercellular adhesion and metastasis. To gain insight into EpCAM function in a physiologic context in vivo, we generated conditional knockout mice with EpCAM-deficient LC and characterized them. Epidermis from these mice contained increased numbers of LC with normal levels of MHC and costimulatory molecules and T-cell-stimulatory activity in vitro. Migration of EpCAM-deficient LC from skin explants was inhibited, but chemotaxis of dissociated LC was not. Correspondingly, the ability of contact allergen-stimulated, EpCAM-deficient LC to exit epidermis in vivo was delayed, and strikingly fewer hapten-bearing LC subsequently accumulated in lymph nodes. Attenuated migration of EpCAM-deficient LC resulted in enhanced contact hypersensitivity responses as previously described in LC-deficient mice. Intravital microscopy revealed reduced translocation and dendrite motility in EpCAM-deficient LC in vivo in contact allergen-treated mice. These results conclusively link EpCAM expression to LC motility/migration and LC migration to immune regulation. EpCAM appears to promote LC migration from epidermis by decreasing LC-keratinocyte adhesion and may modulate intercellular adhesion and cell movement within in epithelia during development and carcinogenesis in an analogous fashion.
Subject(s)
Antigens, Neoplasm/metabolism , Cell Adhesion Molecules/metabolism , Cell Movement , Epidermis/pathology , Langerhans Cells/pathology , Neoplasms/pathology , Animals , Cattle , Cell Adhesion Molecules/deficiency , Cell Movement/drug effects , Cell Shape/drug effects , Chemotaxis/drug effects , Collagen/pharmacology , Dermatitis, Contact/immunology , Dermatitis, Contact/pathology , Epidermis/drug effects , Epithelial Cell Adhesion Molecule , Fluorescent Antibody Technique , Langerhans Cells/drug effects , Mice , Mice, Knockout , Microscopy, Confocal , Neoplasms/metabolism , PhenotypeABSTRACT
Epithelial cell adhesion molecule (EpCAM) (CD326) is a surface glycoprotein expressed by invasive carcinomas and some epithelia. Herein, we report that EpCAM regulates the composition and function of tight junctions (TJ). EpCAM accumulated on the lateral interfaces of human colon carcinoma and normal intestinal epithelial cells but did not co-localize with TJ. Knockdown of EpCAM in T84 and Caco-2 cells using shRNAs led to changes in morphology and adhesiveness. TJ formed readily after EpCAM knockdown; the acquisition of trans-epithelial electroresistance was enhanced, and TJ showed increased resistance to disruption by calcium chelation. Preparative immunoprecipitation demonstrated that EpCAM bound tightly to claudin-7. Co-immunoprecipitation documented associations of EpCAM with claudin-7 and claudin-1 but not claudin-2 or claudin-4. Claudin-1 associated with claudin-7 in co-transfection experiments, and claudin-7 was required for association of claudin-1 with EpCAM. EpCAM knockdown resulted in decreases in claudin-7 and claudin-1 proteins that were reversed with lysosome inhibitors. Immunofluorescence microscopy revealed that claudin-7 and claudin-1 continually trafficked into lysosomes. Although EpCAM knockdown decreased claudin-1 and claudin-7 protein levels overall, accumulations of claudin-1 and claudin-7 in TJ increased. Physical interactions between EpCAM and claudins were required for claudin stabilization. These findings suggest that EpCAM modulates adhesion and TJ function by regulating intracellular localization and degradation of selected claudins.
Subject(s)
Antigens, Neoplasm/metabolism , Cell Adhesion Molecules/metabolism , Claudins/metabolism , Proteolysis , Tight Junctions/metabolism , Antigens, Neoplasm/genetics , Caco-2 Cells , Cell Adhesion/physiology , Cell Adhesion Molecules/genetics , Claudins/genetics , Epithelial Cell Adhesion Molecule , Gene Knockdown Techniques , Humans , Tight Junctions/genetics , TransfectionABSTRACT
In an effort to understand the role of Distal-less 3 (Dlx3) in cutaneous biology and pathophysiology, we generated and characterized a mouse model with epidermal ablation of Dlx3. K14cre;Dlx3(Kin/f) mice exhibited epidermal hyperproliferation and abnormal differentiation of keratinocytes. Results from subsequent analyses revealed cutaneous inflammation that featured accumulation of IL-17-producing CD4(+) T, CD8(+) T, and γδ T cells in the skin and lymph nodes of K14cre;Dlx3(Kin/f) mice. The gene expression signature of K14cre;Dlx3(Kin/f) skin shared features with lesional psoriatic skin, and Dlx3 expression was markedly and selectively decreased in psoriatic skin. Interestingly, cultured Dlx3 null keratinocytes triggered cytokine production that is potentially linked to inflammatory responses in K14cre;Dlx3(Kin/f) mice. Thus, Dlx3 ablation in epidermis is linked to altered epidermal differentiation, barrier development, and IL-17-associated skin inflammation. This model provides a platform that will allow the systematic exploration of the contributions of keratinocytes to cutaneous inflammation.
Subject(s)
Dermatitis/etiology , Homeodomain Proteins/immunology , Interleukin-17/metabolism , Transcription Factors/deficiency , Transcription Factors/immunology , Animals , Cell Differentiation , Cytokines/biosynthesis , Dermatitis/genetics , Dermatitis/immunology , Dermatitis/pathology , Disease Models, Animal , Epidermis/immunology , Epidermis/pathology , Female , Homeodomain Proteins/genetics , Humans , Hyperplasia , Inflammation Mediators/metabolism , Keratinocytes/immunology , Keratinocytes/pathology , Leukocytes/immunology , Leukocytes/pathology , Mice , Mice, Knockout , Pregnancy , Th17 Cells/immunology , Th17 Cells/pathology , Transcription Factors/geneticsABSTRACT
Uptake of Leishmania major by dendritic cells (DCs) results in activation and interleukin (IL)-12 release. Infected DCs efficiently stimulate CD4- and CD8- T cells and vaccinate against leishmaniasis. In contrast, complement receptor 3-dependent phagocytosis of L. major by macrophages (MPhi) leads exclusively to MHC class II-restricted antigen presentation to primed, but not naive, T cells, and no IL-12 production. Herein, we demonstrate that uptake of L. major by DCs required parasite-reactive immunoglobulin (Ig)G and involved FcgammaRI and FcgammaRIII. In vivo, DC infiltration of L. major-infected skin lesions coincided with the appearance of antibodies in sera. Skin of infected B cell-deficient mice and Fcgamma-/- mice contained fewer parasite-infected DCs in vivo. Infected B cell-deficient mice as well as Fcgamma-/- mice (all on the C57BL/6 background) showed similarly increased disease susceptibility as assessed by lesion volumes and parasite burdens. The B cell-deficient mice displayed impaired T cell priming and dramatically reduced IFN-gamma production, and these deficits were normalized by infection with IgG-opsonized parasites. These data demonstrate that DC and MPhi use different receptors to recognize and ingest L. major with different outcomes, and indicate that B cell-derived, parasite-reactive IgG and DC FcgammaRI and FcgammaRIII are essential for optimal development of protective immunity.
Subject(s)
Dendritic Cells/immunology , Leishmania major/immunology , Leishmaniasis, Cutaneous/prevention & control , Receptors, IgG/immunology , Animals , B-Lymphocytes/immunology , Cells, Cultured , Dendritic Cells/parasitology , Immunoglobulin G/immunology , Leishmania major/pathogenicity , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/parasitology , Macrophage-1 Antigen/immunology , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , PhagocytosisABSTRACT
OBJECTIVE: Pericytes/pericyte precursors produce milk fat globule-associated protein with epidermal growth factor and factor VIII-like domains (MFG-E8) in vivo, and this α(v) integrin ligand enhances angiogenesis in tumors and in oxygen-induced retinopathy in mice. Inhibition of MFG-E8 production or function attenuates platelet-derived growth factor-BB (PDGF-BB)-induced migration of pericyte/pericyte precursor-like 10T1/2 cells in vitro. Herein, we describe mechanisms by which MFG-E8 modulates PDGF-BB:PDGF receptor ß (PDGFRß) signaling in 10T1/2 cells. METHODS AND RESULTS: Small interfering RNA depletion of MFG-E8 from 10T1/2 cells or antibody inhibition of MFG-E8 action enhanced PDGF-BB-dependent degradation of PDGFRß and attenuated signaling. Coimmunoprecipitation revealed transient association of MFG-E8 with PDGFRß in PDGF-BB-treated 10T1/2 cells and reduced PDGFRß-focal adhesion kinase association in MFG-E8-depleted cells. Confocal microscopy demonstrated that MFG-E8 binding to 10T1/2 cells was RGD motif and α(v) dependent but PDGF-BB treatment independent, whereas colocalization of MFG-E8 with PDGFRß was enhanced by PDGF-BB. Ubiquitination of PDGFRß was also increased in MFG-E8 small interfering RNA-transfected cells. CONCLUSION: Integrin α(v)-bound MFG-E8 associates with PDGFRß and focal adhesion kinase after PDGF-BB treatment, results in cell surface retention of PDGFRß, delays receptor degradation, potentiates downstream signaling, and enhances migration of 10T1/2 cells. MFG-E8 may promote angiogenesis, in part, via cell autonomous actions on pericytes or pericyte precursors that result in enhanced PDGF-BB:PDGFRß signaling mediated via integrin-growth factor receptor cross-talk.
Subject(s)
Antigens, Surface/metabolism , Embryonic Stem Cells/metabolism , Integrin alphaV/metabolism , Milk Proteins/metabolism , Pericytes/metabolism , Receptor, Platelet-Derived Growth Factor beta/metabolism , Signal Transduction/physiology , Animals , Antigens, Surface/drug effects , Becaplermin , Cell Line , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesion Kinase 1/metabolism , Mice , Mice, Inbred C3H , Milk Proteins/antagonists & inhibitors , Milk Proteins/drug effects , Models, Animal , Pericytes/cytology , Pericytes/drug effects , Phosphorylation/physiology , Platelet-Derived Growth Factor/metabolism , Platelet-Derived Growth Factor/pharmacology , Proto-Oncogene Proteins c-sis , RNA, Small Interfering/pharmacologyABSTRACT
OBJECTIVE: MFG-E8 (also called lactadherin and SED1) is a secreted glycoprotein that has been previously implicated in enhancement of vascular endothelial growth factor-dependent angiogenesis. Major sources of MFG-E8 in vivo and precise mechanisms of MFG-E8 action remain undetermined. The objective of this study was to identify important sources of MFG-E8 in vivo and further elucidate the role(s) of MFG-E8 in the regulation of angiogenesis. METHODS AND RESULTS: We used knockout mice and anti-MFG-E8 antibodies to study MFG-E8 function in vivo. In melanomas and in retinas of mice with oxygen-induced retinopathy, MFG-E8 colocalized with pericytes rather than endothelial cells, and platelet-derived growth factor receptor ß+ pericytes/pericyte precursors purified from tumors contained large amounts of MFG-E8 mRNA. Tumor- and retinopathy-associated angiogenesis was diminished in MFG-E8 knockout mice, and pericyte coverage of neovessels was reduced. Inhibition of MFG-E8 production by 10T1/2 cells (surrogate pericyte/pericyte precursors) using small interfering RNAs and short hairpin RNAs, or inhibition of MFG-E8 action with some anti-MFG-E8 antibodies, selectively attenuated migration in vitro. Significantly, the anti-MFG-E8 antibodies that inhibited 10T1/2 cell migration in vitro also inhibited pathological angiogenesis in vivo. CONCLUSIONS: These studies strongly implicate MFG-E8 in pericyte/pericyte precursor function and indicate that MFG-E8-directed therapeutics may merit further development.
Subject(s)
Antigens, Surface/physiology , Melanoma, Experimental/blood supply , Neovascularization, Pathologic/etiology , Pericytes/metabolism , Animals , Cell Movement , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Milk Proteins , Retinal Diseases/etiologyABSTRACT
A new langerin(+) DC subset has recently been identified in murine dermis (langerin(+) dDC), but the lineage and functional relationships between these cells and langerin(+) epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC allowed viable LC to be easily distinguished from langerin(+) dDC in skin and lymphoid tissue and ex vivo as well. Differential expression of EpCAM and langerin revealed the presence of at least 3 distinct skin DC subsets. We determined that LC and langerin(+) dDC exhibit different migratory capabilities in vitro and repopulate distinct anatomic compartments in skin at different rates after conditional depletion in vivo. Langerin(+) dDC, in contrast to LC, did not require TGFbeta1 for development. Carefully timed gene gun immunization studies designed to take advantage of the distinct repopulation kinetics of langerin(+) dDC and LC revealed that langerin(+) dDC were required for optimal production of beta-galactosidase-specific IgG2a/c and IgG2b in the acute phase. In contrast, immunization via LC-deficient skin resulted in persistent and strikingly reduced IgG1 and enhanced IgG2a Ab production. Our data support the concepts that LC and langerin(+) dDC represent distinct DC subsets that have specialized functions and that LC are important immunoregulatory cells. The presence of at least 3 functionally distinct skin DC subsets may have particular relevance for vaccines that are administered epicutaneously.
Subject(s)
Antigens, Surface/metabolism , Langerhans Cells/metabolism , Lectins, C-Type/metabolism , Mannose-Binding Lectins/metabolism , Animals , Antigens, Surface/immunology , Cell Movement , Female , Kinetics , Langerhans Cells/cytology , Langerhans Cells/immunology , Lectins, C-Type/immunology , Mannose-Binding Lectins/immunology , Mice , Mice, Knockout , Th1 Cells/immunology , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolismABSTRACT
Nucleic acid-based vaccines are effective in infectious disease models but have yielded disappointing results in tumor models when tumor-associated self-antigens are used. Incorporation of helper epitopes from foreign antigens into tumor vaccines might enhance the immunogenicity of DNA vaccines without increasing toxicity. However, generation of fusion constructs encoding both tumor and helper antigens may be difficult, and resulting proteins have unpredictable physical and immunologic properties. Furthermore, simultaneous production of equal amounts of highly immunogenic helper and weakly immunogenic tumor antigens in situ could favor development of responses against the helper antigen rather than the antigen of interest. We assessed the ability of 2 helper antigens (beta-galactosidase or fragment C of tetanus toxin) encoded by one plasmid to augment responses to a self-antigen (lymphoma-associated T-cell receptor) encoded by a separate plasmid after codelivery into skin by gene gun. This approach allowed adjustment of the relative ratios of helper and tumor antigen plasmids to optimize helper effects. Incorporation of threshold (minimally immunogenic) amounts of helper antigen plasmid into a DNA vaccine regimen dramatically increased T cell-dependent protective immunity initiated by plasmid-encoded tumor-associated T-cell receptor antigen. This simple strategy can easily be incorporated into future vaccine trials in experimental animals and possibly in humans.
Subject(s)
Biolistics/methods , Cancer Vaccines/pharmacology , Lymphoma, T-Cell/therapy , Peptide Fragments/genetics , Tetanus Toxin/genetics , Vaccines, DNA/pharmacology , beta-Galactosidase/genetics , Animals , Antibody Formation/immunology , Antigens, Neoplasm/immunology , Cancer Vaccines/immunology , Cell Line, Tumor , Cricetinae , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Female , Kidney/cytology , Lymphoma, T-Cell/immunology , Mice , Mice, Inbred C57BL , Peptide Fragments/immunology , Plasmids/pharmacology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes/immunology , Tetanus Toxin/immunology , Transfection , Vaccines, DNA/immunology , beta-Galactosidase/immunologyABSTRACT
Leishmaniasis is a parasitic disease affecting â¼12 million people. Control of infection (e.g. in C57BL/6 mice) results from IL-12-dependent production of IFNγ by Th1/Tc1 cells. In contrast, BALB/c mice succumb to infection because of preferential Th2-type cytokine induction. Infected dendritic cells (DC) represent important sources of IL-12. Genetically determined differences in DC IL-1α/ß production contribute to disease outcome. Whereas the course of disease was not dramatically altered in IL-1RI(-/-) mice, local administration of IL-1α to infected C57BL/6 mice improved disease outcome. To definitively elucidate the involvement of IL-1 in immunity against leishmaniasis, we now utilized IL-1α/ß-double-deficient C57BL/6 mice. C57BL/6 mice are believed to be a good surrogate model for human, self limited cutaneous leishmaniasis (CL). Leishmania major-infected IL-1α/ß(-/-) mice were resistant to experimental CL comparable to controls. In addition, DC-based vaccination against leishmaniasis in C57BL/6 mice was independent of IL-1. Thus, in Leishmania-resistant C57BL/6 mice, IL-1 signalling is dispensable for protection.
Subject(s)
Interleukin-1/immunology , Leishmania major/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Dendritic Cells/parasitology , Disease Models, Animal , Humans , Interleukin-1alpha/administration & dosage , Interleukin-1alpha/deficiency , Interleukin-1alpha/genetics , Interleukin-1beta/deficiency , Interleukin-1beta/genetics , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/prevention & control , Macrophages/immunology , Macrophages/parasitology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/immunologyABSTRACT
Resistance to leishmaniasis in C57BL/6 mice depends on Th1/Tc1 cells. BALB/c mice preferentially develop Th2 immunity and succumb to infection. We now assessed the role of IL-17 in cutaneous leishmaniasis. During the course of Leishmania major infection, BALB/c CD4 cells and neutrophils produced increased amounts of IL-17 as compared with cells from C57BL/6 mice. This increase was associated with significantly increased IL-23 release from L. major-infected BALB/c dendritic cells (DC), whereas IL-6 and TGF-beta1 production by BALB/c and C57BL/6 DC were comparable. Interestingly, lesion sizes in infected IL-17-deficient BALB/c mice were dramatically smaller and failed to progress as compared with those in control mice. Similar amounts of IL-4, IL-10, and IFN-gamma were produced by T cells from IL-17-deficient mice and control mice consistent with development of Th2-predominant immunity in all animals. Improved disease outcome was associated with decreased CXCL2-accumulation in lesion sites and decreased neutrophil immigration into lesions of infected IL-17-deficient mice confirming prior observations that enhanced neutrophil recruitment contributes to disease susceptibility in BALB/c mice. This study excludes an important facilitating role for IL-17 in Th1/Th2 development in L. major-infected BALB/c mice, and suggests that IL-23 production by L. major-infected DC maintains IL-17(+) cells that influence disease progression via regulation of neutrophil recruitment.
Subject(s)
Genetic Predisposition to Disease , Interleukin-17/physiology , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Cutaneous/immunology , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/parasitology , Dendritic Cells/pathology , Disease Progression , Immunity, Cellular/genetics , Interleukin-17/biosynthesis , Interleukin-17/deficiency , Interleukin-17/genetics , Interleukin-23/metabolism , Leishmania major/immunology , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/immunology , Neutrophils/parasitology , Neutrophils/pathology , Species Specificity , Th2 Cells/immunology , Th2 Cells/parasitology , Th2 Cells/pathology , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
Humans with biallelic inactivating mutations in Epithelial Cell Adhesion Molecule (EpCAM) develop congenital tufting enteropathy (CTE). To gain mechanistic insights regarding EpCAM function in this disorder, we prepared intestinal epithelial cell (IEC) organoids and spheroids. IEC organoids and spheroids were generated from ROSA-CreERT2 EpCAMfl/fl mice. Proliferation, tight junctions, cell polarity and epithelial integrity were assessed in tamoxifen-induced EpCAM-deficient organoids via confocal immunofluorescence microscopy and Western blotting. Olfm4-expressing stem cells were assessed in IEC cells in vitro and in vivo via fluorescence in situ hybridization. To determine if existing drugs could ameliorate effects of EpCAM deficiency in IEC cells, a variety of pharmacologic inhibitors were screened. Deletion of EpCAM resulted in increased apoptosis and attenuated growth of organoids and spheroids. Selected claudins were destabilized and epithelial integrity was severely compromised. Epithelial integrity was improved by treatment with Rho-associated coiled-coil kinase (ROCK) inhibitors without restoration of claudin expression. Correspondingly, enhanced phosphorylation of myosin light chain, a serine/threonine ROCK substrate, was observed in EpCAM-deficient organoids. Strikingly, frequencies of Olfm4-expressing stem cells in EpCAM-deficient IEC cells in vitro and in vivo were decreased. Treatment with ROCK inhibitors increased numbers of stem cells in EpCAM-deficient organoids and spheroids. Thus, EpCAM regulates intestinal epithelial homeostasis via a signaling pathway that includes ROCK.
Subject(s)
Epithelial Cell Adhesion Molecule/metabolism , Epithelial Cells/metabolism , Intestines/cytology , Stem Cells/metabolism , rho-Associated Kinases/metabolism , Animals , Apoptosis/drug effects , Cell Differentiation/drug effects , Cell Polarity/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Claudins/metabolism , Epithelial Cells/drug effects , Gene Silencing , Intestinal Mucosa/metabolism , Mice, Knockout , Myosin Light Chains/metabolism , Organoids/drug effects , Organoids/metabolism , Organoids/ultrastructure , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Stem Cells/drug effects , rho-Associated Kinases/antagonists & inhibitorsABSTRACT
Protective immunity against pathogens such as Leishmania major is mediated by interleukin (IL)-12-dependent Th1-immunity. We have shown previously that skin-dendritic cells (DCs) from both resistant C57BL/6 and susceptible BALB/c mice release IL-12 when infected with L. major, and infected BALB/c DCs effectively vaccinate against leishmaniasis. To determine if cytokines other than IL-12 might influence disease outcome, we surveyed DCs from both strains for production of a variety of cytokines. Skin-DCs produced significantly less IL-1alpha in response to lipopolysaccharide/interferon gamma or L. major when expanded from BALB/c as compared with C57BL/6 mice. In addition, IL-1alpha mRNA accumulation in lymph nodes of L. major-infected BALB/c mice was approximately 3-fold lower than that in C57BL/6 mice. Local injections of IL-1alpha during the first 3 d after infection led to dramatic, persistent reductions in lesion sizes. In L. major-infected BALB/c mice, IL-1alpha administration resulted in increased Th1- and strikingly decreased Th2-cytokine production. IL-1alpha and IL-12 treatments were similarly effective, and IL-1alpha efficacy was strictly IL-12 dependent. These data indicate that transient local administration of IL-1alpha acts in conjunction with IL-12 to influence Th-development in cutaneous leishmaniasis and prevents disease progression in susceptible BALB/c mice, perhaps by enhancing DC-induced Th1-education. Differential production of IL-1 by C57BL/6 and BALB/c mice may provide a partial explanation for the disparate outcomes of infection in these mouse strains.