- Email: [email protected]
The Bone Marrow Is Akin to Skin: HCELL and the Biology of Hematopoietic Stem Cell Homing
The Bone Marrow Is Akin to Skin: HCELL and the Biology of Hematopoietic Stem Cell Homing Robert Sacksteinw
Departments of Dermatology and Medicine, Brigham & Women’s Hospital, Harvard Skin Disease Research Center, Harvard Medical School and wDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
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.
Key words: adult stem cell/hematopoietic stem cell/homing/regenerative therapy/selectin. J Invest Dermatol, J Invest Dermatol 122:1061 –1069, 2004
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 et al, 1998; Orlic et al, 2001; Hess et al, 2002; Hofstetter et al, 2002; Jin et al, 2002; Toma et al, 2002; Hess et al, 2003). 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 et al, 2001; Terada et al, 2002; Ianus et al, 2003; Vassilopoulos et al, 2003), 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 and Dennis, 1996; Wulf et al, 2001; Jiang et al, 2002a, b). MSCs are relatively rare in situ and must be expanded in culture to achieve sufficient numbers, whereas bone marrow natively contains large
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.
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 et al, 2001) and in humans (Stamm et al, 2003; Tse et al, 2003), 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 et al, 2001; Wang et al, 2001). 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.
Copyright r 2004 by The Society for Investigative Dermatology, Inc.
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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, 1957, 1959; Gowans and Steer, 1980). 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 gutassociated lymphoid tissues whereas lymphocytes isolated from peripheral lymph node tended to migrate to those sites (reviewed in Chin et al, 1991). 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 et al, 1983; Rasmussen et al, 1985; Chin et al, 1986; Woodruff et al, 1987; Holzmann et al, 1989). 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 a4b7 (LPAM-1). Had it not been for the fact that L-selectin expression is not restricted to lymphocytes, our understanding of tissuespecific 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, 1993). 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 et al, 1995), 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
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Figure 1 The 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.
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 et al, 1996). 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 Fig 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 Fig 1). This ‘‘multi-step paradigm’’ (Butcher, 1991; Springer, 1994) 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 and Butcher, 2000; Moser and Loetscher, 2001; D’Ambrosio et al, 2003). 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 et al, 1998; Tangemann et al, 1998; Willimann et al, 1998). Neutrophils do not express CCR7, and, therefore, while able to undergo rolling interactions on HEV, they cannot convert these contacts into firm adherence (Warnock et al, 1998). This example emphasizes the fact that the ‘‘address’’ directing cellular trafficking to relevant tissues is created by the combined expression of
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leukocyte and endothelial adhesion molecules and their respective chemokine receptors and chemokines. 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 et al, 1997). 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, a4b7, and LFA-1 (Berlin et al, 1993; Springer, 1994; Alon et al, 1995; DeGrendele et al, 1996; Knorr and Dustin, 1997; Lichtman et al, 1997; Sackstein, 1997; de Chateau et al, 2001). The selectins are a family of integral membrane glycoproteins that function as Ca2 þ -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 et al, 1996; Frenette et al, 1998; Weninger et al, 2000). 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 et al, 1997). 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
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threshold shear level (Finger et al, 1996; Alon et al, 1997; Lawrence et al, 1997). 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 et al, 1988). 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, 1993; Lechleitner et al, 1999; Mikulowska-Mennis et al, 2001; Renkonen et al, 2002). 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 et al, 1993; Asa et al, 1995; Guyer et al, 1996; Spertini et al, 1996; Walcheck et al, 1996), 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 et al, 1995; Li et al, 1996), and PSGL-1 E-selectin ligand activity depends on sialic acid and fucose modifications that are recognized by the rat IgM mAb HECA452 (Duijvestijn et al, 1988), which is directed against a sialyl Lewis X-like epitope (Berg et al, 1991). Inactive and active forms of PSGL-1 are expressed on leukocytes and on hematopoietic progenitor cells (Alon et al, 1994; Laszik et al, 1996; Lichtman et al, 1997). 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 et al, 1990; Berg et al, 1991; Picker et al, 1991). Later biochemical studies showed that the CLA epitope is located on PSGL-1 (Fuhlbrigge et al, 1997), and more recent studies have provided direct evidence that only CLA( þ )PSGL-1 functions as an E-selectin ligand whereas both CLA( þ ) and CLA() glycoforms of PSGL-1 can bind P-selectin (Fuhlbrigge et al, 2002). 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( þ )PSGL-1 on memory T cells engages E-selectin (and P-selectin), which is constitutively expressed on dermal microvasculature (Weninger et al, 2000). 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 et al, 1998;
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Harari et al, 1999, Weninger et al, 2000). 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 et al, 1997; Tamaru et al, 1998; Hickey et al, 1999; Kulidjian et al, 2002; Reinhardt et al, 2003). 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 et al, 1997). 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 et al, 1994; Biedermann et al, 2002).
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 et al, 1996; Frenette et al, 1998). There is increasing evidence these molecules play important roles in trafficking of primitive hematopoietic cells into bone marrow (Frenette et al, 1998; Mazo et al, 1998; Hidalgo et al, 2002; Katayama et al, 2003). Although the identity of the true HSC is debated, for the purposes of this review we will consider CD34 þ cells lacking lineagespecific markers (i.e., CD34 þ /lineage cells) as the representative population of HSC. Human HSC express PSGL-1 and another selectin ligand, HCELL (Oxley and Sackstein, 1994; Sackstein et al, 1997; Dimitroff et al, 2000, 2001a; Sackstein and Dimitroff, 2000). 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 and Sackstein, 1994; Sackstein et al, 1997; Sackstein and Dimitroff, 2000). 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 et al, 1987), 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 and Dailey, 2000; Siegelman et al, 2000; Stoop et al, 2002). The CD44 protein is expressed on essentially all hematopoietic
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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 and Komada, 2000). 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 et al, 2000, 2001a). 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 et al, 2001a, 2001b). 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 þ /CD38/lineage cells) (Sackstein et al, 2001). 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, 1993; Simmons et al, 1994). 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 and Harlan, 1994; Peled et al, 1999a). 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 et al, 1999a). 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 et al, 1996) (and, in mice, P-selectin; Mazo et al, 1998, 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 et al, 1999a; Hidalgo et al, 2002; Katayama et al, 2003). E-selectin ligands and VLA-4 on HSC cooperate and represent the major adhesion molecules mediating homing to bone marrow (Katayama et al, 2003), but the engagement of E-selectin ligands is more important for initiating tethering and primary rolling interactions of human HSC (Peled et al, 1999a). The contribution of VLA-4 in rolling may be a secondary effect, potentiated by the native high levels of the chemokine SDF1 (CXCL12) in the marrow sinusoids coupled with the fact that VCAM-1 is also constitutively expressed on bone marrow endothelium (Mazo et al, 1998). 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
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Figure 2 Molecular 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.
Figure 3 The 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.
immature and mature leukocytes. This restricted cellular distribution and its marked potency for engagement of natively expressed E-selectin on bone marrow endothelium (Dimitroff et al, 2001a) suggest that HCELL functions as the authentic ‘‘bone marrow homing receptor’’ on human HSC (see Fig 2).
ing SDF-1-driven transendothelial migration (Naiyer et al, 1999). 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 et al, 1998; Peled et al, 1999a). 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 et al, 2000; Pillarisetti and Gupta, 2001; Stumm et al, 2002; Kollet et al, 2003), and increased SDF-1 expression recruits human HSC into the livers of immunodeficient mice following hepatic injury (Kollet et al, 2003). 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 Figs 2 and 3). 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 et al, 1999; Fedyk et al, 2001). 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 et al, 1989; Revenga et al, 2000; Alter, 2002; Fernandez Acenero et al, 2003). 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 et al, 2002). 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
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 et al, 2002) 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 et al, 1996; Ma et al, 1998). Postnatally, SDF-1 is produced constitutively in multiple organs by various cells, including endothelial cells, tissue dendritic cells, and bone marrow stromal cells (Pablos et al, 1999; Ponomaryov et al, 2000; Muller et al, 2001). Studies in immune deficient mice have shown that bone marrow homing of human HSC is critically dependent on SDF-1/CXCR4 interactions (Peled et al, 1999b), 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 mediat-
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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 þ cells were seen in the hair follicles, striated muscles, sebaceous glands, and epidermis in host skin 21 d after wounding (Badiavas et al, 2003). 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 et al, 1999; Lagasse et al, 2000; Orlic et al, 2001; Hess et al, 2002, 2003; Mangi et al, 2003). 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 et al, 1999). 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 et al, 1998). 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-selectindeficient states is overcome by the overwhelming concentration(s) of relevant chemoattractants such that the reduced time of exposure is not a critical variable (Ley et al, 1998). 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 (Fig 3). Besides SDF-1, HSC bear receptors for a variety of other chemokines (Broxmeyer et al, 1999; Lee et al, 1999) and chemotactic factors (Mohle et al, 2001), which, if expressed in a tissueor inflammation-specific manner, could synergise with SDF1 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 (Fig 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
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HSC based on expression of HCELL, or, minimally, CLA( þ )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 et al, 1996; Wagers et al, 1998; Lim et al, 1999; Fuhlbrigge et al, 2002), 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. 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 DOI: 10.1111/j.0022-202X.2004.09301.x Manuscript received September 17, 2003; accepted for publication November 13, 2003 Address correspondence to: Robert Sackstein, MD, PhD, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Room 671, Boston, Massachusetts 02115, USA. Email: [email protected]
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Departments of Dermatology and Medicine, Brigham & Women’s Hospital, Harvard Skin Disease Research Center, Harvard Medical School and wDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
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.
Key words: adult stem cell/hematopoietic stem cell/homing/regenerative therapy/selectin. J Invest Dermatol, J Invest Dermatol 122:1061 –1069, 2004
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 et al, 1998; Orlic et al, 2001; Hess et al, 2002; Hofstetter et al, 2002; Jin et al, 2002; Toma et al, 2002; Hess et al, 2003). 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 et al, 2001; Terada et al, 2002; Ianus et al, 2003; Vassilopoulos et al, 2003), 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 and Dennis, 1996; Wulf et al, 2001; Jiang et al, 2002a, b). MSCs are relatively rare in situ and must be expanded in culture to achieve sufficient numbers, whereas bone marrow natively contains large
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.
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 et al, 2001) and in humans (Stamm et al, 2003; Tse et al, 2003), 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 et al, 2001; Wang et al, 2001). 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.
Copyright r 2004 by The Society for Investigative Dermatology, Inc.
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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, 1957, 1959; Gowans and Steer, 1980). 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 gutassociated lymphoid tissues whereas lymphocytes isolated from peripheral lymph node tended to migrate to those sites (reviewed in Chin et al, 1991). 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 et al, 1983; Rasmussen et al, 1985; Chin et al, 1986; Woodruff et al, 1987; Holzmann et al, 1989). 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 a4b7 (LPAM-1). Had it not been for the fact that L-selectin expression is not restricted to lymphocytes, our understanding of tissuespecific 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, 1993). 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 et al, 1995), 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
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Figure 1 The 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.
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 et al, 1996). 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 Fig 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 Fig 1). This ‘‘multi-step paradigm’’ (Butcher, 1991; Springer, 1994) 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 and Butcher, 2000; Moser and Loetscher, 2001; D’Ambrosio et al, 2003). 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 et al, 1998; Tangemann et al, 1998; Willimann et al, 1998). Neutrophils do not express CCR7, and, therefore, while able to undergo rolling interactions on HEV, they cannot convert these contacts into firm adherence (Warnock et al, 1998). This example emphasizes the fact that the ‘‘address’’ directing cellular trafficking to relevant tissues is created by the combined expression of
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leukocyte and endothelial adhesion molecules and their respective chemokine receptors and chemokines. 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 et al, 1997). 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, a4b7, and LFA-1 (Berlin et al, 1993; Springer, 1994; Alon et al, 1995; DeGrendele et al, 1996; Knorr and Dustin, 1997; Lichtman et al, 1997; Sackstein, 1997; de Chateau et al, 2001). The selectins are a family of integral membrane glycoproteins that function as Ca2 þ -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 et al, 1996; Frenette et al, 1998; Weninger et al, 2000). 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 et al, 1997). 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
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threshold shear level (Finger et al, 1996; Alon et al, 1997; Lawrence et al, 1997). 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 et al, 1988). 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, 1993; Lechleitner et al, 1999; Mikulowska-Mennis et al, 2001; Renkonen et al, 2002). 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 et al, 1993; Asa et al, 1995; Guyer et al, 1996; Spertini et al, 1996; Walcheck et al, 1996), 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 et al, 1995; Li et al, 1996), and PSGL-1 E-selectin ligand activity depends on sialic acid and fucose modifications that are recognized by the rat IgM mAb HECA452 (Duijvestijn et al, 1988), which is directed against a sialyl Lewis X-like epitope (Berg et al, 1991). Inactive and active forms of PSGL-1 are expressed on leukocytes and on hematopoietic progenitor cells (Alon et al, 1994; Laszik et al, 1996; Lichtman et al, 1997). 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 et al, 1990; Berg et al, 1991; Picker et al, 1991). Later biochemical studies showed that the CLA epitope is located on PSGL-1 (Fuhlbrigge et al, 1997), and more recent studies have provided direct evidence that only CLA( þ )PSGL-1 functions as an E-selectin ligand whereas both CLA( þ ) and CLA() glycoforms of PSGL-1 can bind P-selectin (Fuhlbrigge et al, 2002). 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( þ )PSGL-1 on memory T cells engages E-selectin (and P-selectin), which is constitutively expressed on dermal microvasculature (Weninger et al, 2000). 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 et al, 1998;
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Harari et al, 1999, Weninger et al, 2000). 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 et al, 1997; Tamaru et al, 1998; Hickey et al, 1999; Kulidjian et al, 2002; Reinhardt et al, 2003). 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 et al, 1997). 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 et al, 1994; Biedermann et al, 2002).
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 et al, 1996; Frenette et al, 1998). There is increasing evidence these molecules play important roles in trafficking of primitive hematopoietic cells into bone marrow (Frenette et al, 1998; Mazo et al, 1998; Hidalgo et al, 2002; Katayama et al, 2003). Although the identity of the true HSC is debated, for the purposes of this review we will consider CD34 þ cells lacking lineagespecific markers (i.e., CD34 þ /lineage cells) as the representative population of HSC. Human HSC express PSGL-1 and another selectin ligand, HCELL (Oxley and Sackstein, 1994; Sackstein et al, 1997; Dimitroff et al, 2000, 2001a; Sackstein and Dimitroff, 2000). 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 and Sackstein, 1994; Sackstein et al, 1997; Sackstein and Dimitroff, 2000). 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 et al, 1987), 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 and Dailey, 2000; Siegelman et al, 2000; Stoop et al, 2002). The CD44 protein is expressed on essentially all hematopoietic
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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 and Komada, 2000). 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 et al, 2000, 2001a). 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 et al, 2001a, 2001b). 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 þ /CD38/lineage cells) (Sackstein et al, 2001). 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, 1993; Simmons et al, 1994). 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 and Harlan, 1994; Peled et al, 1999a). 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 et al, 1999a). 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 et al, 1996) (and, in mice, P-selectin; Mazo et al, 1998, 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 et al, 1999a; Hidalgo et al, 2002; Katayama et al, 2003). E-selectin ligands and VLA-4 on HSC cooperate and represent the major adhesion molecules mediating homing to bone marrow (Katayama et al, 2003), but the engagement of E-selectin ligands is more important for initiating tethering and primary rolling interactions of human HSC (Peled et al, 1999a). The contribution of VLA-4 in rolling may be a secondary effect, potentiated by the native high levels of the chemokine SDF1 (CXCL12) in the marrow sinusoids coupled with the fact that VCAM-1 is also constitutively expressed on bone marrow endothelium (Mazo et al, 1998). 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
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Figure 2 Molecular 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.
Figure 3 The 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.
immature and mature leukocytes. This restricted cellular distribution and its marked potency for engagement of natively expressed E-selectin on bone marrow endothelium (Dimitroff et al, 2001a) suggest that HCELL functions as the authentic ‘‘bone marrow homing receptor’’ on human HSC (see Fig 2).
ing SDF-1-driven transendothelial migration (Naiyer et al, 1999). 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 et al, 1998; Peled et al, 1999a). 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 et al, 2000; Pillarisetti and Gupta, 2001; Stumm et al, 2002; Kollet et al, 2003), and increased SDF-1 expression recruits human HSC into the livers of immunodeficient mice following hepatic injury (Kollet et al, 2003). 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 Figs 2 and 3). 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 et al, 1999; Fedyk et al, 2001). 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 et al, 1989; Revenga et al, 2000; Alter, 2002; Fernandez Acenero et al, 2003). 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 et al, 2002). 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
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 et al, 2002) 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 et al, 1996; Ma et al, 1998). Postnatally, SDF-1 is produced constitutively in multiple organs by various cells, including endothelial cells, tissue dendritic cells, and bone marrow stromal cells (Pablos et al, 1999; Ponomaryov et al, 2000; Muller et al, 2001). Studies in immune deficient mice have shown that bone marrow homing of human HSC is critically dependent on SDF-1/CXCR4 interactions (Peled et al, 1999b), 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 mediat-
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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 þ cells were seen in the hair follicles, striated muscles, sebaceous glands, and epidermis in host skin 21 d after wounding (Badiavas et al, 2003). 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 et al, 1999; Lagasse et al, 2000; Orlic et al, 2001; Hess et al, 2002, 2003; Mangi et al, 2003). 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 et al, 1999). 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 et al, 1998). 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-selectindeficient states is overcome by the overwhelming concentration(s) of relevant chemoattractants such that the reduced time of exposure is not a critical variable (Ley et al, 1998). 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 (Fig 3). Besides SDF-1, HSC bear receptors for a variety of other chemokines (Broxmeyer et al, 1999; Lee et al, 1999) and chemotactic factors (Mohle et al, 2001), which, if expressed in a tissueor inflammation-specific manner, could synergise with SDF1 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 (Fig 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
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HSC based on expression of HCELL, or, minimally, CLA( þ )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 et al, 1996; Wagers et al, 1998; Lim et al, 1999; Fuhlbrigge et al, 2002), 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. 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 DOI: 10.1111/j.0022-202X.2004.09301.x Manuscript received September 17, 2003; accepted for publication November 13, 2003 Address correspondence to: Robert Sackstein, MD, PhD, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Room 671, Boston, Massachusetts 02115, USA. Email: [email protected]
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