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The Bone Marrow Is Akin to Skin: HCELL and the Biology of Hematopoietic Stem Cell Homing

  • Robert Sackstein
    Correspondence
    Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Room 671, Boston, Massachusetts 02115, USA
    Affiliations
    Departments of Dermatology and Medicine, Brigham & Women's Hospital, Harvard Skin Disease Research Center, Harvard Medical School, Boston, Massachusetts, USA

    Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
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      The recent findings that adult stem cells are capable of generating new blood vessels and parenchymal cells within tissues they have colonized has raised immense optimism that these cells may provide functional recovery of damaged organs. The use of adult stem cells for regenerative therapy poses the challenging task of getting these cells into the requisite sites with minimum morbidity and maximum efficiency. Ideally, tissue-specific colonization could be achieved by introducing the stem cells intravascularly and exploiting the native physiologic processes governing cell trafficking. Critical to the success of this approach is the use of stem cells bearing appropriate membrane molecules that mediate homing from vascular to tissue compartments. Hematopoietic stem cells (HSC) express a novel glycoform of CD44 known as hematopoietic cell E-/L-selectin ligand (HCELL). This molecule is the most potent E-selectin ligand natively expressed on any human cell. This article reviews our current understanding of the molecular basis of HSC homing and will describe the fundamental “roll” of HCELL in opening the avenues for efficient HSC trafficking to the bone marrow, the skin and other extramedullary sites.

      Keywords

      Abbreviations

      CLA
      cutaneous lymphocyte antigen
      HCELL
      hematopoietic cell E-/L-selectin ligand
      HEV
      high endothelial venules
      HSC
      Hematopoietic stem cells
      mAb
      monoclonal antibodies
      PSGL-1
      P-selectin glycoprotein ligand-1
      Substantial evidence has accumulated over the past several years that “adult” (i.e., postnatal) stem cells are capable of generating cells not only of their usual somatic lineage but also, depending on where they reside, that of diverse tissues/organs (
      • Azizi S.A.
      • Stokes D.
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      • Prockop D.J.
      Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—similarities to astrocyte grafts.
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      Bone marrow cells regenerate infarcted myocardium.
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      Bone marrow as a source of endothelial cells and NeuN-expressing cells after stroke.
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      Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery.
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      Intracerebral transplantation of mesenchymal stem cells into acid sphingomyelinase-deficient mice delays the onset of neurological abnormalities and extends their life span.
      ;
      • Toma C.
      • Pittenger M.F.
      • Cahill K.S.
      • Byrne B.J.
      • Kessler P.D.
      Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.
      ;
      • Hess D.
      • Li L.
      • Martin M.
      • et al.
      Bone marrow-derived stem cells initiate pancreatic regeneration.
      ). Although it is debated whether this effect is due to stem cell “plasticity” (the capacity to transdifferentiate across typical lineage commitments, even across derivative embryonic layers) or due to fusion and provoked proliferation of resident tissue-specific cells (
      • Wulf G.G.
      • Jackson K.A.
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      Somatic stem cell plasticity: Current evidence and emerging concepts.
      ;
      • Terada N.
      • Hamazaki T.
      • Oka M.
      • et al.
      Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion.
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      In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion.
      ;
      • Vassilopoulos G.
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      • Russell D.W.
      Transplanted bone marrow regenerates liver by cell fusion.
      ), it is indisputable that remarkable new blood vessel and tissue growth can occur. Justifiably, these findings have engendered great hope that adult stem cells will prove useful in curing previously incurable illnesses affecting millions of people across all ages.
      The most plentiful source of adult stem cells in the body is the bone marrow. Two populations of stem cells are marrow residents: the hematopoietic stem cells (HSC) and the non-hematopoietic mesenchymal stem cells (MSCs). Both of these have broad developmental potential and display “plasticity” (
      • Caplan A.I.
      • Dennis J.E.
      Mesenchymal stem cells: Progenitors, progeny, and pathways.
      ;
      • Wulf G.G.
      • Jackson K.A.
      • Goodell M.A.
      Somatic stem cell plasticity: Current evidence and emerging concepts.
      ;
      • Jiang Y.
      • Jahagirdar B.N.
      • Reinhardt R.L.
      • et al.
      Pluripotency of mesenchymal stem cells derived from adult marrow.
      ). MSCs are relatively rare in situ and must be expanded in culture to achieve sufficient numbers, whereas bone marrow natively contains large numbers of HSC. HSC can be obtained readily by direct extraction (harvesting) or by cytokine-stimulated mobilization of the cells into the circulation with subsequent collection by pheresis, and the techniques for collection and processing of highly enriched populations of HSC are clinically routine. Thus, ex vivo expansion of HSC is not required, and these cells are imminently available. A major issue still facing the clinical use of HSC for regenerative therapy, however, is how to achieve sufficient colonization of these cells within the relevant site(s) of tissue damage. Although the direct injection of HSC has already shown promise in recovery of blood supply and function in ischemic heart disease in rodent models (
      • Orlic D.
      • Kajstura J.
      • Chimenti S.
      • et al.
      Bone marrow cells regenerate infarcted myocardium.
      ) and in humans (
      • Stamm C.
      • Westphal B.
      • Kleine H.D.
      • et al.
      Autologous bone-marrow stem-cell transplantation for myocardial regeneration.
      ;
      • Tse H.F.
      • Kwong Y.L.
      • Chan J.K.
      • Lo G.
      • Ho C.L.
      • Lau C.P.
      Angiogenesis in ischemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation,.
      ), this approach is hampered by the need to specifically map affected zones and to perform invasive procedure(s). Clearly, the cost(s) of these intervention(s) and risk(s) of procedure-related morbidity must always be considered. Moreover, in general, direct injection is only feasible for affected organs/tissues with distinct margins and anatomic localization. An alternative method to achieve tissue infiltration is to introduce HSC into the systemic circulation thereby relying on physiologic homing to deliver the cells to the relevant site(s). This approach has already shown promise in preclinical models of stroke and myocardial infarction (
      • Chen J.
      • Li Y.
      • Wang L.
      • Zhang Z.
      • Lu D.
      • Lu M.
      • Chopp M.
      Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats.
      ;
      • Wang J.S.
      • Shum-Tim D.
      • Chedrawy E.
      • Chiu R.C.
      The coronary delivery of marrow stromal cells for myocardial regeneration: Pathophysiologic and therapeutic implications.
      ). In particular, for regeneration of large tissue areas such as skin affected by burns or by autoimmune or congenital/genetic degenerative disease(s), the requisite colonization of stem cells could only be accomplished through a systemic approach.
      To migrate to tissue(s), all circulating cells must first be capable of adhering to vascular endothelium with sufficient strength to overcome the shear forces of blood flow in a process known as “rolling”. This primary adhesive interaction dictates the trafficking of blood-borne cells to any tissue. Accordingly, to attain successful tissue colonization of infused stem cells requires that this process be functionally intact and, preferably, optimized. This review will consider this issue, and will discuss the emerging evidence that a newly discovered E-selectin ligand, hematopoietic cell E-/L-selectin ligand (HCELL), may function as the principal “homing receptor” directing migration of HSC to the bone marrow, the skin and other extramedullary sites of tissue injury/inflammation.

      What Is a “Homing” Receptor?

      The term “homing receptor” is an historical, operational definition, first coined to describe the migration patterns of circulating lymphocytes (
      • Gowans J.L.
      The effect of continuous reinfusion of lymph and lymphocytes on the output of lymphocytes from the thoracic duct of unanaesthetised rats.
      ,
      • Gowans J.L.
      The recirculation of small lymphocytes from blood to lymph in the rat.
      ;
      • Gowans J.L.
      • Steer H.W.
      The function and pathways of lymphocyte recirculation.
      ). Early studies in rodent and sheep models demonstrated that lymphocyte migration to lymphoid tissues was conspicuously non-random: lymphocytes isolated from intestinal lymph tended to migrate through gut-associated lymphoid tissues whereas lymphocytes isolated from peripheral lymph node tended to migrate to those sites (reviewed in
      • Chin Y.H.
      • Sackstein R.
      • Cai J.P.
      Lymphocyte-homing receptors and preferential migration pathways.
      ). Later it became clear that specificity in migration to different lymphoid compartments depended on the ability of circulating lymphocytes to bind to the specialized post-capillary venules of lymph nodes known as “high endothelial venules” (HEV). Monoclonal antibodies (mAb) were raised against lymphocyte membrane glycoproteins that mediated high affinity adhesion to lymphoid organ-specific HEV ligand(s), and these mAb blocked the migration of lymphocytes to peripheral lymph nodes and Peyer's patches (
      • Gallatin W.M.
      • Weissman I.L.
      • Butcher E.C.
      A cell-surface molecule involved in organ-specific homing of lymphocytes.
      ;
      • Rasmussen R.A.
      • Chin Y.H.
      • Woodruff J.J.
      • Easton T.G.
      Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBFLN (A.11) antibody.
      ;
      • Chin Y.H.
      • Rasmussen R.A.
      • Woodruff J.J.
      • Easton T.G.
      A monoclonal anti-HEBFPP antibody with specificity for lymphocyte surface molecules mediating adhesion to Peyer's patch high endothelium of the rat.
      ;
      • Woodruff J.J.
      • Clarke L.M.
      • Chin Y.H.
      Specific cell-adhesion mechanisms determining migration pathways of recirculating lymphocytes.
      ;
      • Holzmann B.
      • McIntyre B.W.
      • Weissman I.L.
      Identification of a murine Peyer's patch-specific lymphocyte homing receptor as an integrin molecule with an alpha chain homologous to human VLA-4 alpha.
      ). These functionally defined homing receptors were then characterized at a molecular level. The biochemical studies revealed that the principal peripheral lymph node homing receptor was a molecule of the selectin class of adhesion molecules named “L-selectin”, whereas the homing receptor for gut-associated lymphoid tissue was another structure of the integrin class called α4β7 (LPAM-1).
      Had it not been for the fact that L-selectin expression is not restricted to lymphocytes, our understanding of tissue-specific homing would have been complete two decades ago. But it was soon clear that all mature leukocytes and, in fact, primitive hematopoietic progenitor cells (but not intermediate differentiating myeloid or lymphoid cells), characteristically express L-selectin (
      • Sackstein R.
      Physiologic migration of lymphocytes to lymph nodes following bone marrow transplantation: Role in immune recovery.
      ). Importantly, in vitro adherence assays under shear conditions showed that L-selectin expressed on neutrophils was capable of directing the binding of these cells to HEV ligands (
      • Lawrence M.B.
      • Berg E.L.
      • Butcher E.C.
      • Springer T.A.
      Rolling of lymphocytes and neutrophils on peripheral node addressin and subsequent arrest on ICAM-1 in shear flow.
      ), yet these cells do not routinely migrate into lymph nodes. These findings raised doubts about the function of L-selectin as the lymph node homing receptor: How could a molecule serve as a homing receptor on one cell and not another? This puzzle was solved with the understanding that physiologic migration of blood-borne cells into tissues requires a cascade of multiple steps.
      Leukocytes typically exit the vasculature at post-capillary venules, where shear stress ranges from 1 to 4 dynes per cm2 (
      • Jones D.A.
      • Smith C.W.
      • McIntire L.V.
      Leucocyte adhesion under flow conditions: Principles important in tissue engineering.
      ). In this state of motion, leukocytes must first make contact along the endothelial surface with adhesive interactions of sufficient strength to overcome the shear forces of blood flow (initial tethering and rolling, Step 1; see Figure 1). During this initial stage, leukocytes are exposed to chemical signals (chemokines, cytokines, and other pro-inflammatory mediators) in the local milieu (Step 2), thereby leading to activation-dependent upregulation of integrin adhesive capabilities resulting in firm arrest (Step 3). Firm arrest is then followed by transmigration (Step 4) (see Figure 1). This “multi-step paradigm” (
      • Butcher E.C.
      Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity.
      ;
      • Springer T.A.
      Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm.
      ) holds that cells capable of homing to any given tissue must possess the capacity to crawl along the endothelium with sufficient time to “taste” the local milieu and must possess the requisite receptors for the environmental chemoattractants such as chemokines, thus leading to activation-induced upregulation of the surface integrins mediating firm adherence. Chemokines are a superfamily of small proteins that function as potent chemotactic agents, some of which have a tissue- and inflammation-specific distribution, and others which are widely distributed (
      • Campbell J.J.
      • Butcher E.C.
      Chemokines in tissue-specific and microenvironment-specific lymphocyte homing.
      ;
      • Moser B.
      • Loetscher P.
      Lymphocyte traffic control by chemokines.
      ;
      • D'Ambrosio D.
      • Panina-Bordignon P.
      • Sinigaglia F.
      Chemokine receptors in inflammation: An overview.
      ). The activation signal(s) for firm adherence are typically mediated by G-protein-coupled chemokine receptors, which have a cell-specific distribution. For the case of lymphocyte homing to peripheral lymph nodes, HEV constitutively express the chemokine SLC (CCL21) that binds the lymphocyte receptor known as CCR7 (
      • Gunn M.D.
      • Tangemann K.
      • Tam C.
      • Cyster J.G.
      • Rosen S.D.
      • Williams L.T.
      A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes.
      ;
      • Tangemann K.
      • Gunn M.D.
      • Giblin P.
      • Rosen S.D.
      A high endothelial cell-derived chemokine induces rapid, efficient, and subset-selective arrest of rolling T lymphocytes on a reconstituted endothelial substrate.
      ;
      • Willimann K.
      • Legler D.F.
      • Loetscher M.
      • et al.
      The chemokine SLC is expressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts activated T cells via CCR7.
      ). Neutrophils do not express CCR7, and, therefore, while able to undergo rolling interactions on HEV, they cannot convert these contacts into firm adherence (
      • Warnock R.A.
      • Askari S.
      • Butcher E.C.
      • von Andrian U.H.
      Molecular mechanisms of lymphocyte homing to peripheral lymph nodes.
      ). This example emphasizes the fact that the “address” directing cellular trafficking to relevant tissues is created by the combined expression of leukocyte and endothelial adhesion molecules and their respective chemokine receptors and chemokines.
      Figure thumbnail gr1
      Figure 1The multi-step model of leukocyte–endothelial interactions. Schematic representation of the multiple steps involved in leukocyte migration from vascular to tissue components. Steps 1a and b together act to “arrest” cells in flow and are mediated principally by selectin–ligand interactions. Chemokine-induced activation (Step 2) of integrin adhesiveness results in firm adherence (Step 3) followed by transmigration (Step 4). See text for details.
      Although the appropriate display of chemokines and chemokine receptors is required for passage through the endothelium, no cellular emigration from the vascular compartment can occur without the initial (Step 1) molecular adhesive interactions that cause decelerative braking (“tethering”) of the flowing cells and organized sustained contact (“rolling”) of these cells against the endothelial surface. These critical contacts are created by leukocyte surface molecules possessing the requisite chemical characteristics to achieve fast on-off bond times with their respective endothelial co-receptors; in the setting of fluid shear forces and consequent cellular torque, this translates into a rolling interaction (
      • Lawrence M.B.
      • Kansas G.S.
      • Kunkel E.J.
      • Ley K.
      Threshold levels of fluid shear promote leukocyte adhesion through selectins (CD62L, P,E).
      ). Thus, Step 1 is the physiologic linchpin for all downstream events and is a key regulatory step in the composition of the infiltrate: only those cells in blood flow that are capable of participating in tethering and rolling interactions will become tissue residents. Indeed, all “homing receptors” are effectors of Step 1 interactions. Thus, despite the selectivity implied by the name, a broader view based on biophysics holds that a homing receptor is, principally, a molecule specialized to allow blood-borne cells to brake, contact and roll along the endothelium at velocities below the prevailing hydrodynamic flow.

      E-Selectin, Cutaneous Lymphocyte Antigen (CLA), and the Human “Skin Homing Receptor”

      A number of well-characterized adhesion molecules serve as mediators of Step 1 rolling interactions: the three members of the selectin family (E-, P-, and L-selectin, also known as CD62E, CD62P, and CD62L, respectively), the “hyaluronate receptor” CD44, and a small subset of the integrin superfamily, principally VLA-4, α4β7, and LFA-1 (
      • Berlin C.
      • Berg E.L.
      • Briskin M.J.
      • et al.
      Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1.
      ;
      • Springer T.A.
      Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm.
      ;
      • Alon R.
      • Kassner P.D.
      • Carr M.W.
      • Finger E.B.
      • Hemler M.E.
      • Springer T.A.
      The integrin VLA-4 supports tethering and rolling in flow on VCAM-1.
      ;
      • DeGrendele H.C.
      • Estess P.
      • Picker L.J.
      • Siegelman M.H.
      CD44 and its ligand hyaluronate mediate rolling under physiologic flow: A novel lymphocyte-endothelial cell primary adhesion pathway.
      ;
      • Knorr R.
      • Dustin M.L.
      The lymphocyte function-associated antigen 1 I domain is a transient binding module for intercellular adhesion molecule (ICAM)-1 and ICAM-3 in hydrodynamic flow.
      ;
      • Lichtman A.H.
      • Ding H.
      • Henault L.
      • Vachino G.
      • Camphausen R.
      • Cumming D.
      • Luscinskas F.W.
      CD45RA-RO+ (memory) but not CD45RA+RO- (naive) T cells roll efficiently on E- and P-selectin and vascular cell adhesion molecule-1 under flow.
      ;
      • Sackstein R.
      Expression of an L-selectin ligand on hematopoietic progenitor cells.
      ;
      • de Chateau M.
      • Chen S.
      • Salas A.
      • Springer T.A.
      Kinetic and mechanical basis of rolling through an integrin and novel Ca2+-dependent rolling and Mg2+-dependent firm adhesion modalities for the alpha 4 beta 7-MAdCAM-1 interaction.
      ). The selectins are a family of integral membrane glycoproteins that function as Ca2+-dependent lectins in binding to carbohydrate determinants expressed on their respective ligands. E- and P-selectin are the “vascular” selectins, which are typically inducible molecules expressed on activated endothelium (and on platelets, for P-selectin), yet are also expressed constitutively on bone marrow and dermal microvasculature (
      • Schweitzer K.M.
      • Drager A.M.
      • van der Valk P.
      • et al.
      Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues.
      ;
      • Frenette P.S.
      • Subbarao S.
      • Mazo I.B.
      • von Andrian U.H.
      • Wagner D.D.
      Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow.
      ;
      • Weninger W.
      • Ulfman L.H.
      • Cheng G.
      • Souchkova N.
      • Quackenbush E.J.
      • Lowe J.B.
      • von Andrian U.H.
      Specialized contributions by alpha(1,3)-fucosyltransferase-IV and FucT-VII during leukocyte rolling in dermal microvessels.
      ). L-selectin is the “leukocyte” selectin and, as mentioned above, is expressed on granulocytes and monocytes, as well as on lymphocytes. The selectins are the most effective molecules in mediating leukocyte tethering and rolling interactions on endothelium in the hydrodynamic conditions of blood flow and are capable of maintaining rolling at higher fluid shear stresses than any other structures (
      • Lawrence M.B.
      • Kansas G.S.
      • Kunkel E.J.
      • Ley K.
      Threshold levels of fluid shear promote leukocyte adhesion through selectins (CD62L, P,E).
      ). Moreover, the selectins bear the unique property of binding optimally to their ligands under physiologic shear conditions, and, in fact, L-selectin–ligand interactions require a threshold shear level (
      • Finger E.B.
      • Puri K.D.
      • Alon R.
      • Lawrence M.B.
      • von Andrian U.H.
      • Springer T.A.
      Adhesion through L-selectin requires a threshold hydrodynamic shear.
      ;
      • Alon R.
      • Chen S.
      • Puri K.D.
      • Finger E.B.
      • Springer T.A.
      The kinetics of l-selectin tethers and the mechanics of selectin-mediated rolling.
      ;
      • Lawrence M.B.
      • Kansas G.S.
      • Kunkel E.J.
      • Ley K.
      Threshold levels of fluid shear promote leukocyte adhesion through selectins (CD62L, P,E).
      ).
      The selectin ligands comprise a diverse group of glycosylated molecules. Lymph node HEV express several glycoprotein L-selectin ligands and each of these is recognized by the mAb MECA79 (
      • Streeter P.R.
      • Rouse B.T.
      • Butcher E.C.
      Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.
      ). MECA 79 antigens (one of which is a specialized glycoform of CD34 with restricted endothelial distribution) are sulfated carbohydrates that are constitutively expressed on peripheral lymph node HEV, but can also be found at sites of inflammation including skin (
      • Sackstein R.
      Physiologic migration of lymphocytes to lymph nodes following bone marrow transplantation: Role in immune recovery.
      ;
      • Lechleitner S.
      • Kunstfeld R.
      • Messeritsch-Fanta C.
      • Wolff K.
      • Petzelbauer P.
      Peripheral lymph node addressins are expressed on skin endothelial cells.
      ;
      • Mikulowska-Mennis A.
      • Xu B.
      • Berberian J.M.
      • Michie S.A.
      Lymphocyte migration to inflamed lacrimal glands is mediated by vascular cell adhesion molecule-1/alpha(4)beta(1) integrin, peripheral node addressin/L-selectin, and lymphocyte function-associated antigen-1 adhesion pathways.
      ;
      • Renkonen J.
      • Tynninen O.
      • Hayry P.
      • Paavonen T.
      • Renkonen R.
      Glycosylation might provide endothelial zip codes for organ-specific leukocyte traffic into inflammatory sites.
      ). The principal leukocyte counter-receptor for the vascular selectins is the glycoprotein known as P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 is a cell surface mucin-like glycoprotein which can serve as a ligand for all three selectins (
      • Sako D.
      • Chang X.J.
      • Barone K.M.
      • et al.
      Expression cloning of a functional glycoprotein ligand for P-selectin.
      ;
      • Asa D.
      • Raycroft L.
      • Ma L.
      • Aeed P.A.
      • Kaytes P.S.
      • Elhammer A.P.
      • Geng J.G.
      The P-selectin glycoprotein ligand functions as a common human leukocyte ligand for P- and E-selectins.
      ;
      • Guyer D.A.
      • Moore K.L.
      • Lynam E.B.
      • Schammel C.M.
      • Rogelj S.
      • McEver R.P.
      • Sklar L.A.
      P-selectin glycoprotein ligand-1 (PSGL-1) is a ligand for L-selectin in neutrophil aggregation.
      ;
      • Spertini O.
      • Cordey A.S.
      • Monai N.
      • Giuffre L.
      • Schapira M.
      P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells.
      ;
      • Walcheck B.
      • Moore K.L.
      • McEver R.P.
      • Kishimoto T.K.
      Neutrophil–neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation of P-selectin in vitro.
      ), however, specialized post-translational modifications on the core PSGL-1 protein backbone are necessary for ligand activity for each of the selectins. In particular, the binding determinants on PSGL-1 for both L- and P-selectin are critically dependent on sulfation of tyrosines in the N-terminal portion of the molecule (
      • Sako D.
      • Comess K.M.
      • Barone K.M.
      • Camphausen R.T.
      • Cumming D.A.
      • Shaw G.D.
      A sulfated peptide segment at the amino terminus of PSGL-1 is critical for P-selectin binding.
      ;
      • Li F.
      • Wilkins P.P.
      • Crawley S.
      • Weinstein J.
      • Cummings R.D.
      • McEver R.P.
      Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin.
      ), and PSGL-1 E-selectin ligand activity depends on sialic acid and fucose modifications that are recognized by the rat IgM mAb HECA452 (
      • Duijvestijn A.M.
      • Horst E.
      • Pals S.T.
      • et al.
      High endothelial differentiation in human lymphoid and inflammatory tissues defined by monoclonal antibody HECA-452.
      ), which is directed against a sialyl Lewis X-like epitope (
      • Berg E.L.
      • Yoshino T.
      • Rott L.S.
      • et al.
      The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1.
      ). Inactive and active forms of PSGL-1 are expressed on leukocytes and on hematopoietic progenitor cells (
      • Alon R.
      • Rossiter H.
      • Wang X.
      • Springer T.A.
      • Kupper T.S.
      Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P and E selectin under physiological flow.
      ;
      • Laszik Z.
      • Jansen P.J.
      • Cummings R.D.
      • Tedder T.F.
      • McEver R.P.
      • Moore K.L.
      P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells.
      ;
      • Lichtman A.H.
      • Ding H.
      • Henault L.
      • Vachino G.
      • Camphausen R.
      • Cumming D.
      • Luscinskas F.W.
      CD45RA-RO+ (memory) but not CD45RA+RO- (naive) T cells roll efficiently on E- and P-selectin and vascular cell adhesion molecule-1 under flow.
      ).
      On human lymphocytes, the expression of the HECA452 determinant on PSGL-1 is known as the CLA. Early studies showed that the majority of lymphocytes in skin display HECA452 reactivity but only a minority of lymphocytes in non-cutaneous sites and in the circulation express this marker. Subsequent studies showed that the CLA determinant was found on a subset of skin-homing memory T cells, and that the CLA epitope itself was involved in binding to E-selectin (
      • Picker L.J.
      • Terstappen L.W.
      • Rott L.S.
      • Streeter P.R.
      • Stein H.
      • Butcher E.C.
      Differential expression of homing-associated adhesion molecules by T cell subsets in man.
      ;
      • Berg E.L.
      • Yoshino T.
      • Rott L.S.
      • et al.
      The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1.
      ;
      • Picker L.J.
      • Kishimoto T.K.
      • Smith C.W.
      • Warnock R.A.
      • Butcher E.C.
      ELAM-1 is an adhesion molecule for skin-homing T cells.
      ). Later biochemical studies showed that the CLA epitope is located on PSGL-1 (
      • Fuhlbrigge R.C.
      • Kieffer J.D.
      • Armerding D.
      • Kupper T.S.
      Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells.
      ), and more recent studies have provided direct evidence that only CLA(+)PSGL-1 functions as an E-selectin ligand whereas both CLA(+) and CLA(-) glycoforms of PSGL-1 can bind P-selectin (
      • Fuhlbrigge R.C.
      • King S.L.
      • Dimitroff C.J.
      • Kupper T.S.
      • Sackstein R.
      Direct real-time observation of E- and P-selectin-mediated rolling on cutaneous lymphocyte-associated antigen immobilized on Western blots.
      ). The association between HECA452-reactivity and lymphocyte trafficking to skin was then operationally linked: just as L-selectin on lymphocytes mediates homing to lymph nodes that constitutively express L-selectin ligands (MECA79 antigens) on HEV, CLA(+)PSGL-1 on memory T cells engages E-selectin (and P-selectin), which is constitutively expressed on dermal microvasculature (
      • Weninger W.
      • Ulfman L.H.
      • Cheng G.
      • Souchkova N.
      • Quackenbush E.J.
      • Lowe J.B.
      • von Andrian U.H.
      Specialized contributions by alpha(1,3)-fucosyltransferase-IV and FucT-VII during leukocyte rolling in dermal microvessels.
      ). Thus, CLA modifications on PSGL-1 promote migration of memory lymphocytes to skin.
      In inflammation, the expression of both E- and P-selectin are upregulated in dermal vessels, and, beyond their constitutive function in promoting steady-state immunosurveillance, studies in mouse models have indicated that these molecules play distinct and overlapping roles in further recruiting T cells to inflamed skin (
      • Tietz W.
      • Allemand Y.
      • Borges E.
      • von Laer D.
      • Hallmann R.
      • Vestweber D.
      • Hamann A.
      CD4+ T cells migrate into inflamed skin only if they express ligands for E- and P-selectin.
      ;
      • Harari O.A.
      • McHale J.F.
      • Marshall D.
      • Ahmed S.
      • Brown D.
      • Askenase P.W.
      • Haskard D.O.
      Endothelial cell E- and P-selectin up-regulation in murine contact sensitivity is prolonged by distinct mechanisms occurring in sequence.
      ). But depending on the inflammatory stimulus and the mouse strain utilized, E-selectin may play a more significant role than P-selectin in mediating dermal leukocytic infiltrates (
      • Ramos C.L.
      • Kunkel E.J.
      • Lawrence M.B.
      • et al.
      Differential effect of E-selectin antibodies on neutrophil rolling and recruitment to inflammatory sites.
      ;
      • Tamaru M.
      • Tomura K.
      • Sakamoto S.
      • Tezuka K.
      • Tamatani T.
      • Narumi S.
      Interleukin-1beta induces tissue- and cell type-specific expression of adhesion molecules in vivo.
      ;
      • Hickey M.J.
      • Kanwar S.
      • McCafferty D.M.
      • Granger D.N.
      • Eppihimer M.J.
      • Kubes P.
      Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen.
      ;
      • Kulidjian A.A.
      • Issekutz A.C.
      • Issekutz T.B.
      Differential role of E-selectin and P-selectin in T lymphocyte migration to cutaneous inflammatory reactions induced by cytokines.
      ;
      • Reinhardt R.L.
      • Bullard D.C.
      • Weaver C.T.
      • Jenkins M.K.
      Preferential accumulation of antigen-specific effector CD4 T cells at an antigen injection site involves CD62E-dependent migration but not local proliferation.
      ). Interestingly, the binding determinant(s) for E-selectin on murine leukocyte membranes is undefined: the only evidence of CLA expression in mice has been obtained on a T cell clone, and then only after extensive antigen-specific activation of that clone (
      • Borges E.
      • Pendl G.
      • Eytner R.
      • Steegmaier M.
      • Zollner O.
      • Vestweber D.
      The binding of T cell-expressed P-selectin glycoprotein ligand-1 to E- and P-selectin is differentially regulated.
      ). In contrast, studies in human skin-SCID mouse chimera models have definitively shown that CLA and E-selectin direct recruitment of human lymphocytes into human skin (
      • Yan H.C.
      • Delisser H.M.
      • Pilewski J.M.
      • et al.
      Leukocyte recruitment into human skin transplanted onto severe combined immunodeficient mice induced by TNF-alpha is dependent on E-selectin.
      ;
      • Biedermann T.
      • Schwarzler C.
      • Lametschwandtner G.
      • et al.
      Targeting CLA/E-selectin interactions prevents CCR4-mediated recruitment of human Th2 memory cells to human skin in vivo.
      ).

      The Selectin Ligands of HSC: PSGL-1 and HCELL

      As stated above, the bone marrow microvasculature is like that of skin in that it displays constitutive expression of E- and P-selectin (
      • Schweitzer K.M.
      • Drager A.M.
      • van der Valk P.
      • et al.
      Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues.
      ;
      • Frenette P.S.
      • Subbarao S.
      • Mazo I.B.
      • von Andrian U.H.
      • Wagner D.D.
      Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow.
      ). There is increasing evidence these molecules play important roles in trafficking of primitive hematopoietic cells into bone marrow (
      • Frenette P.S.
      • Subbarao S.
      • Mazo I.B.
      • von Andrian U.H.
      • Wagner D.D.
      Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow.
      ;
      • Mazo I.B.
      • Gutierrez-Ramos J.C.
      • Frenette P.S.
      • Hynes R.O.
      • Wagner D.D.
      • von Andrian U.H.
      Hematopoietic progenitor cell rolling in bone marrow microvessels: Parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.
      ;
      • Hidalgo A.
      • Weiss L.A.
      • Frenette P.S.
      Functional selectin ligands mediating human CD34(+) cell interactions with bone marrow endothelium are enhanced postnatally.
      ;
      • Katayama Y.
      • Hidalgo A.
      • Furie B.C.
      • Vestweber D.
      • Furie B.
      • Frenette P.S.
      PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and {alpha}4 integrin.
      ). Although the identity of the true HSC is debated, for the purposes of this review we will consider CD34+ cells lacking lineage-specific markers (i.e., CD34+/lineage cells) as the representative population of HSC. Human HSC express PSGL-1 and another selectin ligand, HCELL (
      • Oxley S.M.
      • Sackstein R.
      Detection of an L-selectin ligand on a hematopoietic progenitor cell line.
      ;
      • Sackstein R.
      • Fu L.
      • Allen K.L.
      A hematopoietic cell L-selectin ligand exhibits sulfate-independent binding activity.
      ;
      • Dimitroff C.J.
      • Lee J.Y.
      • Fuhlbrigge R.C.
      • Sackstein R.
      A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells.
      ,
      • Dimitroff C.J.
      • Lee J.Y.
      • Rafii S.
      • Fuhlbrigge R.C.
      • Sackstein R.
      CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.
      ;
      • Sackstein R.
      • Dimitroff C.J.
      A hematopoietic cell L-selectin ligand that is distinct from PSGL-1 and displays N-glycan-dependent binding activity.
      ). HCELL was initially identified operationally as an L-selectin ligand, and was distinguished from all other L-selectin ligands by a number of biochemical features: (1) Sulfation-independent binding activity; (2) functional resistance to O-sialoglycoprotease digestion; (3) absence of MECA79 antigens; and (4) L-selectin binding determinants that were expressed on N-glycans rather than O-glycans (
      • Oxley S.M.
      • Sackstein R.
      Detection of an L-selectin ligand on a hematopoietic progenitor cell line.
      ;
      • Sackstein R.
      • Fu L.
      • Allen K.L.
      A hematopoietic cell L-selectin ligand exhibits sulfate-independent binding activity.
      ;
      • Sackstein R.
      • Dimitroff C.J.
      A hematopoietic cell L-selectin ligand that is distinct from PSGL-1 and displays N-glycan-dependent binding activity.
      ). Although initially considered to be a “novel” selectin ligand by the above biochemical criteria, mass spectrometry subsequently revealed that HCELL is not novel per se: it is a glycoform of a well-recognized integral membrane glycoprotein, CD44, that expresses the CLA epitope (i.e., is recognized by mAb HECA452). In contrast to PSGL-1 that displays CLA on O-glycans, however, the CLA determinant(s) and the E-/L-selectin binding sites of HCELL are on N-glycans.
      Historically, CD44 has been known best for its role in binding extracellular matrix elements, principally hyaluronic acid (for which it is known as the “hyaluronic acid receptor”), but also fibronectin, collagen, and chondroitin sulfate. Notably, at one time, CD44 was thought to be the “lymph node homing receptor” (
      • Jalkanen S.
      • Wu N.
      • Bargatze R.F.
      • Butcher E.C.
      Human lymphocyte and lymphoma homing receptors.
      ), but it was subsequently determined that it plays little role in lymphocyte trafficking to lymphoid tissues but a key role in lymphocyte migration to inflammatory sites (
      • Rigby S.
      • Dailey M.O.
      Traffic of L-selectin-negative T cells to sites of inflammation.
      ;
      • Siegelman M.H.
      • Stanescu D.
      • Estess P.
      The CD44-initiated pathway of T-cell extravasation uses VLA-4 but not LFA-1 for firm adhesion.
      ;
      • Stoop R.
      • Gal I.
      • Glant T.T.
      • McNeish J.D.
      • Mikecz K.
      Trafficking of CD44-deficient murine lymphocytes under normal and inflammatory conditions.
      ). The CD44 protein is expressed on essentially all hematopoietic cells, as well as on epithelial cells, fibroblasts, and endothelial cells, and its structure has been extensively characterized. CD44 is an extremely heterogenous and pleiotropic molecule, due to alternative splicing of ten encoding exons and a variety of post-translational modifications (reviewed in
      • Deguchi T.
      • Komada Y.
      Homing-associated cell adhesion molecule (H-CAM/CD44) on human CD34+ hematopoietic progenitor cells.
      ). But the HCELL phenotype is present predominantly on the “standard” CD44 isoform (core m.w. ∼37,000) and migrates as a glycoprotein of ∼98,000 m.w. on SDS-PAGE (
      • Dimitroff C.J.
      • Lee J.Y.
      • Fuhlbrigge R.C.
      • Sackstein R.
      A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells.
      ,
      • Dimitroff C.J.
      • Lee J.Y.
      • Rafii S.
      • Fuhlbrigge R.C.
      • Sackstein R.
      CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.
      ).
      PSGL-1 expressed on human HSC bears the CLA epitope(s) and functions as a ligand for all three selectins. But HCELL is ∼5-fold more avid an L-selectin ligand than PSGL-1, and it expresses more potent E-selectin ligand activity over a far wider shear range than PSGL-1 (
      • Dimitroff C.J.
      • Lee J.Y.
      • Rafii S.
      • Fuhlbrigge R.C.
      • Sackstein R.
      CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.
      ,
      • Dimitroff C.J.
      • Lee J.Y.
      • Schor K.S.
      • Sandmaier B.M.
      • Sackstein R.
      Differential L-selectin binding activities of human hematopoietic cell L-selectin ligands, HCELL and PSGL-1.
      ). Thus, HCELL is the principal E- and L-selectin ligand of human HSC. Furthermore, whereas PSGL-1 is expressed on immature and mature myeloid and lymphoid cells, HCELL expression is characteristic only of primitive hematopoietic cells, especially the earliest subset of HSC lacking CD38 expression (CD34+/CD38-/lineage cells) (
      • Sackstein R.
      • Dimitroff C.J.
      • Lee J.Y.
      • Fuhlbrigge R.C.
      • Parmar K.
      • Mauch P.M.
      • Sandmaier B.M.
      Homing and hematopoiesis: HCELL is the principal E-selectin and L-selectin ligand of human hematopoietic stem cells.
      ).
      Besides PSGL-1 and HCELL, two other molecules capable of serving as homing receptors are also characteristically expressed on primitive human HSC, VLA-4, and LFA-1 (
      • Sackstein R.
      Physiologic migration of lymphocytes to lymph nodes following bone marrow transplantation: Role in immune recovery.
      ;
      • Simmons P.J.
      • Zannettino A.
      • Gronthos S.
      • Leavesley D.
      Potential adhesion mechanisms for localisation of haemopoietic progenitors to bone marrow stroma.
      ). VLA-4 and LFA-1 can serve in overlapping roles in rolling or firm adherence, depending on the avidity state of the molecules following cellular encounters with activating chemokines or cytokines (
      • Carlos T.M.
      • Harlan J.M.
      Leukocyte–endothelial adhesion molecules.
      ;
      • Peled A.
      • Grabovsky V.
      • Habler L.
      • et al.
      The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow.
      ). LFA-1, however, is typically specialized to function solely in firm adherence, and, depending on the cell type, VLA-4 function may also be restricted: e.g., in human HSC, VLA-4 functions mostly in tethering—not rolling—and only at low shear stress under resting conditions, and activation leads to only transient rolling with rapid transition to firm adherence (
      • Peled A.
      • Grabovsky V.
      • Habler L.
      • et al.
      The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow.
      ). In this regard, initial “braking” and rolling of hematopoietic progenitors on bone marrow endothelium most likely occurs through selectin receptor–ligand interactions involving PSGL-1 and HCELL with constitutively expressed E-selectin (
      • Schweitzer K.M.
      • Drager A.M.
      • van der Valk P.
      • et al.
      Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues.
      ) (and, in mice, P-selectin;
      • Mazo I.B.
      • Gutierrez-Ramos J.C.
      • Frenette P.S.
      • Hynes R.O.
      • Wagner D.D.
      • von Andrian U.H.
      Hematopoietic progenitor cell rolling in bone marrow microvessels: Parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.
      , but constitutive P-selectin expression in marrow sinusoids has not been shown in humans). Results of recent studies using human HSC in a xenogeneic (mouse) repopulation model underscores the importance of selectins in the homing of human HSC to the marrow (
      • Peled A.
      • Grabovsky V.
      • Habler L.
      • et al.
      The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow.
      ;
      • Hidalgo A.
      • Weiss L.A.
      • Frenette P.S.
      Functional selectin ligands mediating human CD34(+) cell interactions with bone marrow endothelium are enhanced postnatally.
      ;
      • Katayama Y.
      • Hidalgo A.
      • Furie B.C.
      • Vestweber D.
      • Furie B.
      • Frenette P.S.
      PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and {alpha}4 integrin.
      ). E-selectin ligands and VLA-4 on HSC cooperate and represent the major adhesion molecules mediating homing to bone marrow (
      • Katayama Y.
      • Hidalgo A.
      • Furie B.C.
      • Vestweber D.
      • Furie B.
      • Frenette P.S.
      PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and {alpha}4 integrin.
      ), but the engagement of E-selectin ligands is more important for initiating tethering and primary rolling interactions of human HSC (
      • Peled A.
      • Grabovsky V.
      • Habler L.
      • et al.
      The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow.
      ). The contribution of VLA-4 in rolling may be a secondary effect, potentiated by the native high levels of the chemokine SDF-1 (CXCL12) in the marrow sinusoids coupled with the fact that VCAM-1 is also constitutively expressed on bone marrow endothelium (
      • Mazo I.B.
      • Gutierrez-Ramos J.C.
      • Frenette P.S.
      • Hynes R.O.
      • Wagner D.D.
      • von Andrian U.H.
      Hematopoietic progenitor cell rolling in bone marrow microvessels: Parallel contributions by endothelial selectins and vascular cell adhesion molecule 1.
      ). Of all the homing receptors expressed on HSC, HCELL is unique in that it is expressed solely on HSC, whereas PSGL-1, VLA-4, and LFA-1 are each characteristically expressed on a variety of immature and mature leukocytes. This restricted cellular distribution and its marked potency for engagement of natively expressed E-selectin on bone marrow endothelium (
      • Dimitroff C.J.
      • Lee J.Y.
      • Rafii S.
      • Fuhlbrigge R.C.
      • Sackstein R.
      CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.
      ) suggest that HCELL functions as the authentic “bone marrow homing receptor” on human HSC (see Figure 2).
      Figure thumbnail gr2
      Figure 2Molecular components of hematopoietic stem cell (HSC)–bone marrow endothelium interactions. The relevant molecules on the HSC membrane (dots) and marrow endothelial membrane (dashes) that form receptor–ligand interactions crucial for HSC homing to bone marrow are depicted. See text for details.

      SDF-1 and E-Selectin: The “Roll” of HCELL in Directing Homing of HSC to Bone Marrow and Extramedullary Sites

      For all cells emigrating the vasculature, the process of transmigration is coordinated with the delivery of cells to appropriate parenchymal microenvironments. Chemokines are presented on the luminal surface at the tips of endothelial microvilli, a topography that optimizes the recognition of these molecules by cells in flow (
      • Middleton J.
      • Patterson A.M.
      • Gardner L.
      • Schmutz C.
      • Ashton B.A.
      Leukocyte extravasation: Chemokine transport and presentation by the endothelium.
      ) and promotes the transition from rolling to firm adherence. Following firm adherence, the transendothelial movement and intraparenchymal migration are directed by chemokine gradients. The major chemokine involved in recruitment of HSC is SDF-1 (also known as CXCL12), which binds to its cognate receptor CXCR4. The SDF-1/CXCR4 axis is non-redundant, i.e., an exception among the chemokine family in that one receptor binds one chemokine and vice versa. This axis plays a critical role in development, and mice made deficient in either CXCR4 or SDF-1 die prenatally with deficits in hematopoiesis (due to lack of HSC migration from the fetal liver to bone marrow during embryogenesis), and neural and cardiac development (
      • Nagasawa T.
      • Hirota S.
      • Tachibana K.
      • et al.
      Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1.
      ;
      • Ma Q.
      • Jones D.
      • Borghesani P.R.
      • et al.
      Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice.
      ). Postnatally, SDF-1 is produced constitutively in multiple organs by various cells, including endothelial cells, tissue dendritic cells, and bone marrow stromal cells (
      • Pablos J.L.
      • Amara A.
      • Bouloc A.
      • et al.
      Stromal-cell derived factor is expressed by dendritic cells and endothelium in human skin.
      ;
      • Ponomaryov T.
      • Peled A.
      • Petit I.
      • et al.
      Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function.
      ;
      • Muller A.
      • Homey B.
      • Soto H.
      • et al.
      Involvement of chemokine receptors in breast cancer metastasis.
      ). Studies in immune deficient mice have shown that bone marrow homing of human HSC is critically dependent on SDF-1/CXCR4 interactions (
      • Peled A.
      • Petit I.
      • Kollet O.
      • et al.
      Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.
      ), and in vitro transwell studies of human HSC and human bone marrow endothlelial cells have revealed a critical role for endothelial E-selectin expression in mediating SDF-1-driven transendothelial migration (
      • Naiyer A.J.
      • Jo D.Y.
      • Ahn J.
      • et al.
      Stromal derived factor-1-induced chemokinesis of cord blood CD34(+) cells (long-term culture-initiating cells) through endothelial cells is mediated by E-selectin.
      ). Prior to transmigration, the E-selectin–ligand interactions create optimal slow rolling velocities that enable high efficiency recognition of chemokines such as SDF-1 (
      • Ley K.
      • Allietta M.
      • Bullard D.C.
      • Morgan S.
      Importance of E-selectin for firm leukocyte adhesion in vivo.
      ;
      • Peled A.
      • Grabovsky V.
      • Habler L.
      • et al.
      The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow.
      ). Recent reports have revealed concomitant upregulation of endothelial and parenchymal SDF-1 expression in extramedullary sites following chemical injury, viral inflammation, antigenic challenge, irradiation, and ischemia (
      • Gonzalo J.A.
      • Lloyd C.M.
      • Peled A.
      • Delaney T.
      • Coyle A.J.
      • Gutierrez-Ramos J.C.
      Critical involvement of the chemotactic axis CXCR4/stromal cell-derived factor-1 alpha in the inflammatory component of allergic airway disease.
      ;
      • Pillarisetti K.
      • Gupta S.K.
      Cloning and relative expression analysis of rat stromal cell derived factor-1 (SDF-1): SDF-1 alpha mRNA is selectively induced in rat model of myocardial infarction.
      ;
      • Stumm R.K.
      • Rummel J.
      • Junker V.
      • et al.
      A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: Isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia.
      ;
      • Kollet O.
      • Shivtiel S.
      • Chen Y.Q.
      • et al.
      HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver.
      ), and increased SDF-1 expression recruits human HSC into the livers of immunodeficient mice following hepatic injury (
      • Kollet O.
      • Shivtiel S.
      • Chen Y.Q.
      • et al.
      HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver.
      ). Collectively, these data indicate that whether expressed constitutively or induced by local injury, SDF-1 and E-selectin play essential roles in regulating the trafficking and localization of HSC (see Figure 2 and Figure 3).
      Figure thumbnail gr3
      Figure 3The multi-step model of hematopoietic stem cell (HSC) migration. Schematic model of HSC migration to extramedullary tissues, showing the central roles of endothelial E-selectin and of chemokine SDF-1 in mediating transmigration and seeding of HSC within tissue microenvironment(s). See text for details.
      Besides sharing the distinction of constitutive E-selectin expression, the microvascular endothelium of skin and bone marrow each constitutively express SDF-1, and, just as with hematopoietic stroma, Langerhans cells and dermal fibroblasts also constitutively express SDF-1 (
      • Pablos J.L.
      • Amara A.
      • Bouloc A.
      • et al.
      Stromal-cell derived factor is expressed by dendritic cells and endothelium in human skin.
      ;
      • Fedyk E.R.
      • Jones D.
      • Critchley H.O.
      • Phipps R.P.
      • Blieden T.M.
      • Springer T.A.
      Expression of stromal-derived factor-1 is decreased by IL-1 and TNF and in dermal wound healing.
      ). Commensurate with these similarities between skin and bone marrow, cutaneous hematopoiesis has been observed in a variety of conditions of bone marrow failure, myeloproliferative disorders, and hematopoietic stress (
      • Bowden J.B.
      • Hebert A.A.
      • Rapini R.P.
      Dermal hematopoiesis in neonates: Report of five cases.
      ;
      • Revenga F.
      • Horndler C.
      • Aguilar C.
      • Paricio J.
      Cutaneous extramedullary hematopoiesis.
      ;
      • Alter B.P.
      Bone marrow failure syndromes in children.
      ;
      • Fernandez Acerno M.J.
      • Borbujo J.
      • Villanueva C.
      • Penalver J.
      Extramedullary hematopoiesis in an adult.
      ). Although cutaneous hematopoiesis is a rare event, the fact that blood cells are produced at all within the inhospitable hematopoietic microenvironment of the skin is clear evidence that the native expression of E-selectin and SDF-1 are sufficient for recruitment of HSC to skin. Furthermore, skin “chimerism” (i.e., identification of epithelial cells of donor genotype) has been described following clinical (myeloablative) HSC transplantation (
      • Korbling M.
      • Katz R.L.
      • Khanna A.
      • et al.
      Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells.
      ). The finding that mesodermally derived HSC of the donor contribute to epithelial lineages in the host offers further evidence of the “plasticity” of stem cells. The low level of skin chimerism observed following clinical HSC transplantation may reflect the fact that these observations have been made in atraumatic, intact skin. In an excisional wound model in mice having previously received bone marrow transplantation from syngeneic green fluorescent protein (GFP)-expressing transgenics, significant numbers of differentiated GFP+ cells were seen in the hair follicles, striated muscles, sebaceous glands, and epidermis in host skin 21 d after wounding (
      • Badiavas E.V.
      • Abedi M.
      • Butmarc J.
      • Falanga V.
      • Quesenberry P.
      Participation of bone marrow derived cells in cutaneous wound healing.
      ). These and other published data clearly show that the relative level of tissue regeneration ascribable to effect(s) of colonized stem cells (hematopoietic or mesenchymal) are correlated with the degree of tissue injury, as if adequate “space” must be present for engraftment and cell proliferation induced by infiltrating stem cells (
      • Petersen B.E.
      • Bowen W.C.
      • Patrene K.D.
      • et al.
      Bone marrow as a potential source of hepatic oval cells.
      ;
      • Lagasse E.
      • Connors H.
      • Al-Dhalimy M.
      • et al.
      Purified hematopoietic stem cells can differentiate into hepatocytes in vivo.
      ;
      • Orlic D.
      • Kajstura J.
      • Chimenti S.
      • et al.
      Bone marrow cells regenerate infarcted myocardium.
      ;
      • Hess D.C.
      • Hill W.D.
      • Martin-Studdard A.
      • Carroll J.
      • Brailer J.
      • Carothers J.
      Bone marrow as a source of endothelial cells and NeuN-expressing cells after stroke.
      ,
      • Hess D.
      • Li L.
      • Martin M.
      • et al.
      Bone marrow-derived stem cells initiate pancreatic regeneration.
      ;
      • Mangi A.A.
      • Noiseux N.
      • Kong D.
      • He H.
      • Rezvani M.
      • Ingwall J.S.
      • Dzau V.J.
      Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts.
      ).
      Although rodent models suggest that both E- and P-selectin contribute to directing leukocyte migration to inflammatory sites, the primary endothelial selectin expressed in primates at sites of inflammation is E-selectin (
      • Yao L.
      • Setiadi H.
      • Xia L.
      • Laszik Z.
      • Taylor F.B.
      • McEver R.P.
      Divergent inducible expression of P-selectin and E-selectin in mice and primates.
      ). Moreover, as stated above, there are important mouse strain-specific differences in the relative expression of E- and P-selectin during inflammatory processes. Based on these differences, results obtained from murine models may not correlate with clinical conditions, and studies of human cell trafficking in mice may only reveal the minimum contribution(s) of E-selectin in the biology of homing. Despite these limitations, studies in E-selectin knockout mice have definitively shown that leukocyte recruitment is dependent on slow rolling interactions mediated by E-selectin under physiologic conditions (
      • Ley K.
      • Allietta M.
      • Bullard D.C.
      • Morgan S.
      Importance of E-selectin for firm leukocyte adhesion in vivo.
      ). The E-selectin-mediated slow rolling transit time allows for optimal responses to the local milieu. In those experimental situations where chemoattractants/chemokines are presented in high concentrations (as by injection into a localized site), the increased transit time in E-selectin-deficient states is overcome by the overwhelming concentration(s) of relevant chemoattractants such that the reduced time of exposure is not a critical variable (
      • Ley K.
      • Allietta M.
      • Bullard D.C.
      • Morgan S.
      Importance of E-selectin for firm leukocyte adhesion in vivo.
      ).
      A number of important facts have emerged from existing studies of human HSC homing in vivo using mouse–human chimera models as surrogates of human physiology. From these investigations, a model emerges in which recruitment and retention of HSC to a given site are dependent on recognition of SDF-1 gradients. Because SDF-1 expression is characteristic of many tissues and is further upregulated at sites of inflammation/ischemia, the limiting feature of achieving HSC migration to/colonization of extramedullary sites is the capacity of the circulating cells to undergo the key Step 1 HSC–endothelial adhesive interactions essential to recognition of this chemokine Figure 3. Besides SDF-1, HSC bear receptors for a variety of other chemokines (
      • Broxmeyer H.E.
      • Kim C.H.
      • Cooper S.H.
      • Hangoc G.
      • Hromas R.
      • Pelus L.M.
      Effects of CC, CXC, C, and CX3C chemokines on proliferation of myeloid progenitor cells, and insights into SDF-1-induced chemotaxis of progenitors.
      ;
      • Lee B.
      • Ratajczak J.
      • Doms R.W.
      • Gewirtz A.M.
      • Ratajczak M.Z.
      Coreceptor/chemokine receptor expression on human hematopoietic cells: Biological implications for human immunodeficiency virus-type 1 infection.
      ) and chemotactic factors (
      • Mohle R.
      • Bautz F.
      • Denzlinger C.
      • Kanz L.
      Transendothelial migration of hematopoietic progenitor cells. Role of chemotactic factors.
      ), which, if expressed in a tissue- or inflammation-specific manner, could synergise with SDF-1 in directing HSC migration to appropriate target sites. The selective recruitment of HSC would critically hinge, in any case, on the capacity of the cells in blood flow to undergo decelerating tethering and rolling interactions and “see” the local deposits of these chemoattractants Figure 3.
      In order to optimize this process for infusion-based stem cell therapies, HSC should be enriched for their capacity to bind E-selectin. Accordingly, it would be important to select HSC based on expression of HCELL, or, minimally, CLA(+)PSGL-1. Beyond this, in vitro manipulations to increase the level of expression or the E-selectin ligand activity of HCELL and/or PSGL-1 could be utilized to enhance HSC homing capabilities. For T cells, methodologies to increase CLA expression by transfection of relevant glycosyltransferases and by use of specific growth conditions and cytokines have been described (
      • Knibbs R.N.
      • Craig R.A.
      • Natsuka S.
      • Chang A.
      • Cameron M.
      • Lowe J.B.
      • Stoolman L.M.
      The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells.
      ;
      • Wagers A.J.
      • Waters C.M.
      • Stoolman L.M.
      • Kansas G.S.
      Interleukin 12 and interleukin 4 control T cell adhesion to endothelial selectins through opposite effects on alpha1, 3-fucosyltransferase VII gene expression.
      ;
      • Lim Y.C.
      • Henault L.
      • Wagers A.J.
      • Kansas G.S.
      • Luscinskas F.W.
      • Lichtman A.H.
      Expression of functional selectin ligands on Th cells is differentially regulated by IL-12 and IL-4.
      ;
      • Fuhlbrigge R.C.
      • King S.L.
      • Dimitroff C.J.
      • Kupper T.S.
      • Sackstein R.
      Direct real-time observation of E- and P-selectin-mediated rolling on cutaneous lymphocyte-associated antigen immobilized on Western blots.
      ), and each of these increases skin homing of these cells. These data suggest that similar approaches could be used to increase the expression of relevant sialofucosyl modifications rendering amplified E-selectin ligand activity on HSC. A more direct and immediate approach is to develop reagents such as “activating” mAb that can markedly enhance the capacity of HCELL and/or PSGL-1 to bind E-selectin. This strategy does not require cell culture or extensive ex vivo manipulation(s) of HSC, and is currently under intense study by our laboratory. All of the aforementioned strategies could be combined with angiographically guided infusions of the HSC into the relevant vascular bed(s) to attain a “first pass” effect and minimize migration to non-target tissues. Clearly, validating the promise of the clinical application of stem cell therapy for repair of devastating tissue injury will depend on the rapid development of methodologies to efficiently and safely place these cells where they are most needed. Although the ideal approach to achieve this end is not yet known, there is no doubt that manipulation of the molecular mediators of the physiologic trafficking of stem cells will literally open the “avenues” to safe and effective tissue regeneration.

      ACKNOWLEDGMENTS

      This work is supported by the United States National Institutes of Health (Heart, Lung, and Blood Institute and National Cancer Institute) grants RO1-HL73714, RO1-HL60528, and RO1-CA84156. I thank Dr. Robert C. Fuhlbrigge, Mr Derek Cain, and Ms Angie Kafka for assistance in manuscript preparation, and my patients, alive and deceased, for their courage and inspiration.
      This work was presented at the 52nd Annual Montagna Symposium on the Biology of Skin, “Stem Cells in Skin”, June 13–17, 2003, Snowmass Village, Colorado, USA

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