In contrast to its usual pattern of inducible expression, E-selectin is constitutively expressed on dermal and BM microvascular endothelium, indicating that selective tropism(s) of circulating cells to these tissues is regulated by expression of cell-specific E-selectin ligands. The goal of this study was to identify E-selectin ligands expressed on human HPCs that could function in mediating HPC homing to BM. Earlier studies of E-selectin ligands expressed on mature human leukocytes showed that expression of the HECA-452 epitope(s) on lymphocyte PSGL-1 and on neutrophil L-selectin correlates with E-selectin binding capability (Picker et al. 1991b
; Fuhlbrigge et al. 1997
; Zöllner et al. 1997
). Similarly, in the mouse, HECA-452 decoration of the neutrophil E-selectin ligand, ESL-1, was found to predict E-selectin ligand activity (Zöllner and Vestweber 1996
). Since expression of HECA-452 epitope(s) is functionally correlated with E-selectin ligand activity of glycoproteins expressed on mature leukocytes, we sought to determine whether HECA-452–reactive glycoproteins on human HPCs could function as E-selectin ligands.
To analyze the E-selectin ligand activity of human hematopoietic cells, we performed parallel-plate flow chamber studies of CHO-E cell tethering and rolling on hematopoietic cells lines and of hematopoietic cell rolling on naturally expressed E-selectin on IL-1α–stimulated primary BM endothelial cultures. The data presented here show that KG1a and HL60 cells, and not RPMI-8402 or K562 cells, possess E-selectin ligand activity. The E-selectin–mediated adhesive interactions between these hematopoietic cell lines and CHO-E cells parallel results of binding studies using primary BM endothelial cells, demonstrating the utility and fidelity of CHO-E as a cellular reagent to detect and analyze E-selectin ligands. Consistent with results from these binding studies, Western blot analysis of HECA-452 epitope(s) revealed that KG1a and HL60 membrane proteins display HECA-452–reactive glycoproteins, whereas RPMI-8402 and K562 membrane proteins do not. The major HECA-452–reactive membrane glycoprotein(s) of HL60 cells consist of the monomer (140 kD) form of PSGL-1, however, KG1a cells possess multiple HECA-452–reactive glycoproteins at MW of 74, 100, 120, and 190 kD in addition to the 140-kD and 220-kD (dimer) PSGL-1 bands.
Using the hydrodynamic shear-based blot rolling assay to investigate the E-selectin ligand activity of HL60 and KG1a membrane glycoproteins, we show here that the HECA-452–reactive PSGL-1 expressed on these cells functions as an E-selectin ligand. Additionally, with the exception of the 74-kD glycoprotein, HECA-452–reactive glycoproteins each exhibit E-selectin ligand activity on KG1a cells at 100, 120, and 190 kD. The absence of CHO-E–binding interactions by the 74-kD HECA-452 reactive protein indicates that the presence of the HECA-452 epitope does not, in itself, confer E-selectin ligand activity. Moreover, the observations that HECA-452 staining of immunoblots did not block CHO-E cell binding to relevant glycoproteins, and that HECA-452 incubation of KG1a cells and HL60 cells did not inhibit rolling on BMEC or CHO-E binding to the cells, indicate that the HECA-452 epitope is not the direct binding determinant for E-selectin.
The data obtained from blot rolling assays of Hermes-1–immunoprecipitated KG1a membrane proteins demonstrate that the E-selectin ligand activity at 100 and 190 kD is solely due to CD44, and that CD44 is the major contributor of the E-selectin ligand activity at 120 kD. The residual E-selectin ligand activity observed at 120 kD following exhaustive Hermes-1 immunoprecipitation of KG1a lysates may reflect the minor contribution of a non–CD44 E-selectin ligand, or, less likely, of a CD44 isoform lacking the Hermes-1 epitope. The E-selectin–binding, HECA-452–reactive glycoform of CD44 is also expressed on the CD34+ progenitor cell subset of normal human BM mononuclear cells and on blasts from some de novo human leukemias, but is not present on more mature (lineage+) BM cells, including those enriched for subpopulations consisting of monocytes, granulocytes, and lymphocytes.
Blot rolling assays of HECA-452 immunoblots of KG1a membrane protein show that the cumulative E-selectin ligand activity of HECA-452–reactive CD44 (100-, 120-, and 190-kD bands) was markedly greater than that represented by PSGL-1 (140 and 220 kD), and reveal that the predominant E-selectin ligand of human hematopoietic cells is the 100-kD CD44 band. This 100-kD form of CD44 corresponds to the “standard” hematopoietic form of CD44 (Stamenkovic et al. 1991
), and it is the only form immunoprecipitated from normal human BM cells ( A). Since this molecule is also an L-selectin ligand (Dimitroff et al. 2000
), we now designate this structure as hematopoietic cell E-/L-selectin ligand (HCELL). However, unlike PSGL-1, which binds all three selectins, HCELL is not a ligand for P-selectin. Compared with PSGL-1, in flow chamber assays (a) HCELL/CD44 E-selectin binding was significantly higher at 2.8 dynes/cm2
and functioned over a wider range of shear stress, and (b) HCELL/CD44 expression conferred a greater capacity of human hematopoietic cells to roll on E-selectin naturally expressed on BM microvasculature under physiologic shear stress conditions. These data show that HCELL is a major glycoprotein ligand on human HPCs and suggest that engagement of HPCs with E-selectin at a physiologic postcapillary venule shear stress level is mediated preferentially by HCELL.
Sialidase and fucosidase treatment each abrogated E-selectin–binding activity of CD44, highlighting the importance of sialic acid and fucose modifications for creation of the selectin binding determinant. Elimination of ligand activity following N
-glycanase digestion of KG1a membrane protein indicates that these critical sialofucosylated modifications are displayed on N
-glycosylations, whereas the insensitivity to OSGE digestion suggests that the relevant glycosylations for E-selectin binding of HCELL are not clustered on extensively O
-glycosylated peptide regions (Steininger et al. 1992
). Our data showing that CD44 lacking HECA-452 did not possess E-selectin ligand activity is consistent with results from prior studies showing that HECA-452–nonreactive CD44 from human tonsils does not exhibit E-selectin ligand activity (Berg et al. 1991
). Thus, CD44, a molecule principally known for its capacity to bind extracellular matrix molecules such as hyaluronic acid (HA) (Aruffo et al. 1990
), requires critical sialylated and fucosylated carbohydrate modifications to function as HCELL. Of interest, sialylation of CD44 has been shown to inhibit binding to HA (Skelton et al. 1998
; Katoh et al. 1999
), which may explain the finding that though human CD34+ BM cells characteristically express high levels of CD44 (Kansas et al. 1990
; Peled et al. 1999
), an unexpectedly small fraction of these cells bind to HA (Peled et al. 1999
Previous studies have shown that human HPCs possess E-selectin ligand(s) (Naiyer et al. 1999
; Peled et al. 1999
; Greenberg et al. 2000
) and that human HPC interactions with human BMECs under shear conditions are mediated principally by E-selectin receptor/ligand interactions (Schweitzer et al. 1996
; Rood et al. 2000
), but the human HPC E-selectin ligand(s) have not been identified heretofore. In the mouse, intravital microscopy studies have demonstrated that E-selectin, which is constitutively expressed on murine BM microvasculature in vivo,
can mediate HPC rolling interactions and that adhesive interactions between very late antigen (VLA)-4 expressed on HPCs and BM endothelium vascular cell adhesion molecule (VCAM)-1 also mediate rolling interactions (Mazo et al. 1998
). However, even though IL-1α stimulation of primary BMEC cultures markedly upregulates the expression of VCAM-1 (Rafii et al. 1994
) and despite high VLA-4 expression on KG1a and HL60 cells (Oxley and Sackstein 1994
), rolling of KG1a or HL60 cells on primary BMECs did not utilize the VLA-4/VCAM-1 receptor–ligand pair, as evidenced by the complete abrogation of rolling interactions by anti–E-selectin mAb treatment alone. This finding is consistent with prior reports showing that VCAM-1 in the absence of SDF-1 is incapable of supporting CD34+
adhesive interactions on BM endothelium under flow (Peled et al. 1999
; Schweitzer et al. 1996
; Rood et al. 2000
), further highlighting the importance of E-selectin in this process.
The broad expression of the triselectin ligand PSGL-1 (and of VLA-4) among both immature and mature nucleated hematopoietic cells raises the possibility that selectivity in human HPC homing to BM may involve expression of non–PSGL-1 E-selectin ligands, such as HCELL, on human HPC. In this regard, the fact that HCELL expression is uniquely restricted to CD34+
hematopoietic cells indicates that this CD44 glycoform may impart specificity for progenitor cell rolling on BM microvasculature. Interestingly, though binding to HA could contribute to CD34+
rolling interactions on BM endothelium (DeGrendele et al. 1996
; Peled et al. 1999
), the rather extensive distribution of CD44 among both immature and mature human BM cells (Kansas et al. 1990
) and the fact that human CD34+
cell rolling on endothelium is not inhibited by antibodies that block binding to HA and is inhibited by chelation of divalent ions (which does not affect CD44–HA binding) (Peled et al. 1999
), indicates that CD44–HA interactions are neither unique nor critical to HPC rolling on BM endothelium. Other studies have provided various independent lines of evidence showing that CD44 may play a role in HPC homing to BM (Khaldoyanidi et al. 1996
; Zoller et al. 1998
), in tissue distribution of myeloid progenitors (Schmits et al. 1997
), and in engraftment of transplanted hematopoietic progenitor cells (Sandmaier et al. 1990
; Watanabe et al. 1998
). Thus, whereas L-selectin is operationally known as the “lymph node homing receptor” and CLA is known as a “skin homing receptor,” the data reported here are consistent with a role for HCELL as a “bone marrow homing receptor.” Our findings expand the current knowledge of the structural biology of E-selectin ligands, bestow a new perspective on ligands facilitating HPC trafficking to E-selectin-expressing tissue microvessel beds, and have broad implications exploiting HCELL as a biological surrogate for positive selection of HPCs with improved engraftment potential.