ALBERT

Description: Stem cells are capable of extensive self-renewal in the absence of differentiation. The maintenance of this undifferentiated state occurs despite the fact that this cell is exposed to a milieu that is rich in a variety of growth and differentiation factors. A unifying feature of such hematopoietic factors is that they mediate their effects through the phosphorylation of tyrosine residues by various cellular kinases. Therefore, one mechanism that might inhibit such differentiation signals in the self-renewing stem cell is the dephosphorylation of tyrosine residues by protein tyrosine phosphatases (PTPs). We have thus investigated the types of tyrosine phosphatases expressed by murine embryonic lin(lo)CD34hiSca(hi) hematopoietic progenitor cells by using a consensus polymerase chain reaction (PCR) approach. Although many known tyrosine phosphatases were detected using this method, a novel PTP related to the previously described PTP PEST type enzymes, murine PTP PEP and murine/human PTP PEST, was also observed. Cloning of the full-length cDNA encoding this enzyme showed that it was indeed a novel new member of this family, with an amino terminal tyrosine phosphatase domain followed by a region rich in serine, threonine, and proline. The carboxy terminus of this novel PTP contained a short sequence that was homologous to a region of the murine PTP PEP that was involved with nuclear localization. Bacterial expression of the phosphatase domain showed that this enzyme could efficiently dephosphorylate tyrosines in vitro. Analysis of the expression of the novel nuclear PTP by quantitative PCR showed that the transcript disappeared as the lin(lo)CD34hiSca(hi) cells differentiated in the presence of interleukin-1, interleukin-3, erythropoietin, and granulocyte- macrophage colony-stimulating factor. In agreement with its potential role in the hematopoietic progenitor cell, this novel PTP was expressed at a barely detectable level in a very limited subset of adult tissues. However, analysis of several murine hematopoietic progenitor cell lines, but not of a differentiated T-cell line, showed a high level of expression of the novel PTP. These data suggest that this novel phosphatase may play a critical role in the maintenance of the undifferentiated state of the hematopoietic stem cell.

Description: The processes of angiogenesis and hematopoiesis require a high degree of coordination during embryogenesis. Whereas much is understood about the development of the vascular system in avian embryos, little information has been attained in mammals, predominantly because there are no specific markers for either blood vessels or hematopoietic cells in any developing mammalian system. We have recently shown that murine CD34 (mCD34) is expressed on the vascular endothelium in all organs and tissues of the adult mouse as well as on a small percentage of presumably hematopoietic stem cells in the bone marrow and fetal liver. Here we show that mCD34 is also expressed on the endothelium of blood vessels and on a subset of hematopoietic-like cells throughout murine development. mCD34 is first observed on the yolk sac endothelium of day 7.5 embryos and on a subset of hematopoietic cells within these yolk sacs. mCD34 expression is maintained on vessels and hematopoietic cells in all organs and tissues throughout embryogenesis. In addition, mCD34 is localized on growth conelike filopodial processes that appear at the budding edge of newly sprouted capillaries. Double staining of capillaries for mCD34 and laminin shows that these growth conelike processes seem to be free of laminin, whereas the formed capillaries seem to be coated with this extracellular matrix protein. Analysis of vessels in developing brain shows that these filopodial processes seem to be directed toward the ventricular epithelium, a previously described site of vascular endothelial growth factor synthesis. Finally, we show that the vascular structures of developing murine embryoid bodies also express mCD34. These data suggest that mCD34 is a useful marker for the analysis of the development of the blood vascular system in murine embryos.

Description: Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.

Description: Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.

Description: Embryonic hematopoiesis is initiated in part in the blood islands of the yolk sac. Previous confocal microscopic analysis has shown that the CD34 antigen, a mucin-like cell surface glycoprotein that is expressed by hematopoietic progenitors and all endothelial cells of the adult and embryo, is also found on a subset of luminal hematopoietic-like cells in the yolk sac blood islands as well as on the vascular endothelium lining these early hematopoietic locations. We show here that, as in all other hematopoietic sites thus far examined, immunoaffinity- purified CD34+ nonadherent cells from murine yolk sacs contain the vast majority of erythroid and myeloid progenitor cell colony forming activity. To examine the developmental interactions between these CD34+ hematopoietic progenitor cells of the yolk sac and the CD34+ yolk sac endothelium, we have immunaffinity-purified adherent endothelial cells from day 10.5 yolk sacs using CD34 antiserum and produced cell lines by transformation with a retrovirus expressing the polyoma middle T antigen. Analysis of these cell lines for CD34, von Willebrand's factor, FLK 1 and FLT 1 expression, and capillary growth in Matrigel indicates that they appear to be endothelial cells, consistent with their original phenotype in vivo. Coculture of yolk sac CD34+ hematopoietic cells on these endothelial cell lines results in up to a 60-fold increase in total hematopoietic cell number after approximately 8 days. Analysis of these expanded hematopoietic cells showed that the majority were of the monocyte/macrophage lineage. In addition, examination of the cultures showed the rapid formation of numerous cobblestone areas, a previously described morphologic entity thought to be representative of early pluripotential stem cells. Scrutiny of the ability of these endothelial cell lines to expand committed progenitor cells showed up to a sixfold increase in erythroid and myeloid colony- forming cells after 3 to 6 days in culture, consistent with the notion that these embryonic endothelial cells mediate the expansion of these precursor cells. Polymerase chain reaction analyses showed that most of the cell lines produce FLK-2/FLT-3 ligand, stem cell factor, macrophage colony-stimulating factor, leukemia-inhibitory factor, and interleukin- 6 (IL-6), whereas there is a generally low or not measurable production of granulocyte colony-stimulating factor, granulocyte-macrophage colony- stimulating factor, IL-1, IL-3, transforming growth factor beta-1, erythropoietin, or thrombopoietin. The output of mature hematopoietic cells from these cocultures can be modified to include an erythroid population by the addition of exogenous erythropoietin. These data suggest that endothelial cell lines derived form the yolk sac provide an appropriate hematopoietic environment for the expansion and differentiation of yolk sac progenitor cells into at least the myeloid and erythroid lineages.

Description: During the inflammatory response, granulocytes and other leukocytes adhere to and emigrate from small venules. Before firm attachment, leukocytes are observed rolling slowly along the endothelium in venules of most tissues accessible to intravital microscopy. The molecular mechanism underlying this early type of leukocyte-endothelial interaction is unknown. Leukocyte rolling was investigated in venules (diameter, 40 microns) of the exposed rat mesentery. Micro-infusion of a recombinant soluble chimera (LEC-IgG) of the murine homing receptor lectin-like cell adhesion molecule 1 (LEC-CAM 1; gp90MEL) into individual venules reduced the number of rolling leukocytes by 89% +/- 2% (mean +/- SEM, n = 20 venules), while a similar CD4 chimera (CD4- IgG) had no effect (inhibition 14% +/- 7%, n = 25). Rolling was also greatly reduced by a polyclonal serum against LEC-CAM 1 (inhibition 84% +/- 3%, n = 35); preimmune serum was ineffective (11% +/- 13% inhibition, n = 28). These findings indicate that LEC-CAM 1 mediates the adhesive interaction underlying leukocyte rolling and thus may play an important role in inflammation and in pathologic conditions involving leukocytes.

Description: During the inflammatory response, granulocytes and other leukocytes adhere to and emigrate from small venules. Before firm attachment, leukocytes are observed rolling slowly along the endothelium in venules of most tissues accessible to intravital microscopy. The molecular mechanism underlying this early type of leukocyte-endothelial interaction is unknown. Leukocyte rolling was investigated in venules (diameter, 40 microns) of the exposed rat mesentery. Micro-infusion of a recombinant soluble chimera (LEC-IgG) of the murine homing receptor lectin-like cell adhesion molecule 1 (LEC-CAM 1; gp90MEL) into individual venules reduced the number of rolling leukocytes by 89% +/- 2% (mean +/- SEM, n = 20 venules), while a similar CD4 chimera (CD4- IgG) had no effect (inhibition 14% +/- 7%, n = 25). Rolling was also greatly reduced by a polyclonal serum against LEC-CAM 1 (inhibition 84% +/- 3%, n = 35); preimmune serum was ineffective (11% +/- 13% inhibition, n = 28). These findings indicate that LEC-CAM 1 mediates the adhesive interaction underlying leukocyte rolling and thus may play an important role in inflammation and in pathologic conditions involving leukocytes.

Description: Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34- null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34- null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.