Flow Cytometry
Tan, C., et al (2020). "Extracellular CIRP Induces Inflammation in Alveolar Type II Cells via TREM-1" Front Cell Dev Biol 8: 579157. PubMed
Extracellular cold-inducible RNA-binding protein (eCIRP) induces acute lung injury (ALI) in sepsis. Triggering receptor expressed on myeloid cells-1 (TREM-1) serves as a receptor for eCIRP to induce inflammation in macrophages and neutrophils. The effect of eCIRP on alveolar epithelial cells (AECs) remains unknown. We hypothesize that eCIRP induces inflammation in AECs through TREM-1. AECs were isolated from C57BL/6 mice and freshly isolated AECs were characterized as alveolar type II (ATII) cells by staining AECs with EpCAM, surfactant protein-C (SP-C), and T1 alpha (T1alpha) antibodies. AECs were stimulated with recombinant murine (rm) CIRP and assessed for TREM-1 by flow cytometry. ATII cells from WT and TREM-1(-/-) mice were stimulated with rmCIRP and assessed for interleukin-6 (IL-6) and chemokine (C-X-C motif) ligand 2 (CXCL2) in the culture supernatants. ATII cells from WT mice were pretreated with vehicle (PBS), M3 (TREM-1 antagonist), and LP17 (TREM-1 antagonist) and then after stimulating the cells with rmCIRP, IL-6 and CXCL2 levels in the culture supernatants were assessed. All of the freshly isolated AECs were ATII cells as they expressed EpCAM and SP-C, but not T1alpha (ATI cells marker). Treatment of ATII cells with rmCIRP significantly increased TREM-1 expression by 56% compared to PBS-treated ATII cells. Stimulation of WT ATII cells with rmCIRP increased IL-6 and CXCL2 expression, while the expression of IL-6 and CXCL2 in TREM-1(-/-) ATII cells were reduced by 14 and 23%, respectively. Pretreatment of ATII cells with M3 and LP17 significantly decreased the expression of IL-6 by 30 and 47%, respectively, and CXCL2 by 27 and 34%, respectively, compared to vehicle treated ATII cells after stimulation with rmCIRP. Thus, eCIRP induces inflammation in ATII cells via TREM-1 which implicates a novel pathophysiology of eCIRP-induced ALI and directs a possible therapeutic approach targeting eCIRP-TREM-1 interaction to attenuate ALI.
Immunofluorescence
Peranzoni, E., et al (2018). "Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-PD-1 treatment" Proc Natl Acad Sci U S A 115(17): E4041-E4050. PubMed
In a large proportion of cancer patients, CD8 T cells are excluded from the vicinity of cancer cells. The inability of CD8 T cells to reach tumor cells is considered an important mechanism of resistance to cancer immunotherapy. We show that, in human lung squamous-cell carcinomas, exclusion of CD8 T cells from tumor islets is correlated with a poor clinical outcome and with a low lymphocyte motility, as assessed by dynamic imaging on fresh tumor slices. In the tumor stroma, macrophages mediate lymphocyte trapping by forming long-lasting interactions with CD8 T cells. Using a mouse tumor model with well-defined stromal and tumor cell areas, macrophages were depleted with PLX3397, an inhibitor of colony-stimulating factor-1 receptor (CSF-1R). Our results reveal that a CSF-1R blockade enhances CD8 T cell migration and infiltration into tumor islets. Although this treatment alone has minor effects on tumor growth, its combination with anti-PD-1 therapy further increases the accumulation of CD8 T cells in close contact with malignant cells and delays tumor progression. These data suggest that the reduction of macrophage-mediated T cell exclusion increases tumor surveillance by CD8 T cells and renders tumors more responsive to anti-PD-1 treatment.
Western Blot
Kuan, II, et al (2017). "EpEX/EpCAM and Oct4 or Klf4 alone are sufficient to generate induced pluripotent stem cells through STAT3 and HIF2alpha" Sci Rep 7: 41852. PubMed
Epithelial cell adhesion molecule (EpCAM) was reported to be cleaved into extracellular domain of EpCAM (EpEX) and intracellular domain of EpCAM (EpICD). We previously reported that EpCAM serves as a potent stem cell marker which is highly and selectively expressed by undifferentiated rather than differentiated hESC. However, the functional role of EpCAM remains elusive. Here, we found that EpEX and EpCAM enhance the efficiency of OSKM reprogramming. Interestingly, Oct4 or Klf4 alone, but not Sox2, can successfully reprogram fibroblasts into iPSCs with EpEX and EpCAM. Moreover, EpEX and EpCAM trigger reprogramming via activation of STAT3, which leads to the nuclear-translocation of HIF2alpha. This study reveals the importance of a novel EpEX/EpCAM-STAT3-HIF2alpha signal in the reprogramming process, and uncovers a new means of triggering reprogramming by delivery of soluble and transmembrane proteins.
Flow Cytometry
Baik, S., et al (2016). "Relb acts downstream of medullary thymic epithelial stem cells and is essential for the emergence of RANK(+) medullary epithelial progenitors" Eur J Immunol 46(4): 857-862. PubMed
Thymic epithelial cells (TECs) provide essential signals for alphabetaT-cell development, and medullary TECs (mTECs) control T-cell tolerance through both negative selection and Foxp3(+) regulatory T (Treg) cell development. Although heterogeneity within the mTEC compartment is well studied, the molecular regulators of specific stages of mTEC development are still poorly understood. Given the importance of the RANK-RANKL axis in thymus medulla formation, we have used RANK Venus reporter mice to analyze the ontogeny of RANK(+) TECs during development and correlated RANK expression with mTEC stem cells defined by SSEA-1. In addition, we have investigated how requirements for the key regulators Foxn1 and Relb map to specific stages of mTEC development. Here, we show SSEA-1(+) mTEC stem cells emerge prior to RANK expression and are present in both nude and Relb(-/-) mice, providing direct evidence that mTEC lineage specification occurs independently of Foxn1 and Relb. In contrast, we show that Relb is necessary for the effective production of downstream RANK(+) mTEC progenitors. Collectively, our work defines stage-specific requirements for critical TEC regulators during medulla development, including the timing of Relb dependency, and provides new information on mechanisms controlling mTEC specification.
Immunohistochemistry (frozen)
Tanaka, M., et al (2009). "Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: drastic change of EpCAM expression during liver development" Mech Dev 126(8-9): 665-676. PubMed
Hepatoblasts are hepatic progenitor cells that expand and give rise to either hepatocyte or cholangiocytes during liver development. We previously reported that delta-like 1 homolog (DLK1) is expressed in the mouse liver primordium at embryonic day (E) 10.5 and that DLK1(+) cells in E14.5 liver contain high proliferative and bipotential hepatoblasts. While the expression of epithelial cell adhesion molecule (EpCAM) in hepatic stem/progenitor cells has been reported, its expression profile at an early stage of liver development remains unknown. In this study, we show that EpCAM is expressed in mouse liver bud at E9.5 and that EpCAM(+)DLK1(+) hepatoblasts form hepatic cords at the early stage of hepatogenesis. DLK1(+) cells of E11.5 liver were fractionated into EpCAM(+) and EpCAM(-) cells; one forth of EpCAM(+)DLK1(+) cells formed a colony in vitro whereas EpCAM(-)DLK1(+) cells rarely did it. Moreover, EpCAM(+)DLK1(+) cells contained cells capable of forming a large colony, indicating that EpCAM(+)DLK1(+) cells in E11.5 liver contain early hepatoblasts with high proliferation potential. Interestingly, EpCAM expression in hepatoblasts was dramatically reduced along with liver development and the colony-forming capacities of both EpCAM(+)DLK1(+) and EpCAM(-)DLK1(+) cells were comparable in E14.5 liver. It strongly suggested that most of mouse hepatoblasts are losing EpCAM expression at this stage. Moreover, we provide evidence that EpCAM(+)DLK1(+) cells in E11.5 liver contain extrahepatic bile duct cells as well as hepatoblasts, while EpCAM(-)DLK1(+) cells contain mesothelial cell precursors. Thus, the expression of EpCAM and DLK1 suggests the developmental pathways of mouse liver progenitors.
Immunohistochemistry (frozen)
Trzpis, M., et al (2007). "Spatial and temporal expression patterns of the epithelial cell adhesion molecule (EpCAM/EGP-2) in developing and adult kidneys" Nephron Exp Nephrol 107(4): e119-131. PubMed
BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is expressed by most epithelia and is involved in processes fundamental for morphogenesis, including cell-cell adhesion, proliferation, differentiation, and migration. Previously, a role for EpCAM in pancreatic morphogenesis was confirmed in vitro. Furthermore, changes in the EpCAM expression pattern were found in developing lung and thymus and in the regenerating liver. Therefore, EpCAM was proposed to be a morphoregulatory molecule. METHODS: Using immunohistochemistry, the expression pattern of human and murine homologues of EpCAM was characterized in adult and embryonic kidneys from humans and human-EpCAM (hEpCAM)-transgenic mice. RESULTS: EpCAM expression was found in the ureteric bud throughout nephrogenesis. EpCAM was not expressed in the metanephric mesenchyme. In comma- and S-shaped bodies, both metanephric mesenchyme derived structures, EpCAM expression appeared by E13.5. In adult kidneys, most epithelia expressed varying levels of EpCAM, as confirmed by double staining for human EpCAM and segment-specific nephron markers. Podocytes were EpCAM negative. At the cellular level, the EpCAM expression shifted from apical in embryonic to basolateral in adult kidneys. CONCLUSIONS: The spatiotemporal expression pattern of EpCAM changes during nephrogenesis. In the adult kidney, the expression varies markedly along the nephron. These data provide a basis for further studies on EpCAM in developing and adult kidneys.
Immunohistochemistry (frozen)
Farr, A., et al (1991). "Epithelial heterogeneity in the murine thymus: a cell surface glycoprotein expressed by subcapsular and medullary epithelium" J Histochem Cytochem 39(5): 645-653. PubMed
A monoclonal antibody (MAb), G8.8, was raised against glycoconjugates isolated from a cloned line of murine medullary thymic epithelial cells. Flow cytometric analysis of the reactivity of this MAb with cultured thymic epithelium demonstrated that the ligand was expressed on the cell surface. Immunohistochemical examination of normal murine thymus revealed labeling of cells in the subcapsular and medullary areas. Immunoelectron microscopy revealed surface labeling restricted to cells possessing ultrastructural features of epithelium (desmosomes, tonofilaments, and cytoplasmic cysts). During thymic ontogeny, G8.8+ cells predominated in fetal development at the earliest time point examined (Day 14 of gestation). There was an expansion of the cortical epithelial component so that by Day 18 cortical and medullary compartments could be clearly distinguished. Immunoprecipitation of radioiodinated thymic stroma with MAb G8.8 detected a molecule with an apparent Mr of approximately 38 KD under non-reducing conditions. When reduced, the apparent Mr was slightly increased (42 KD). This MAb also exhibited reactivity with gut and epidermal epithelium and some tubular epithelium in the kidney, but did not react with epithelial parenchymal cells of the liver.
