To assess the ability of mature myeloid cell–derived CD44 to bind E-selectin, we induced the differentiation of the 32D cell line with G-CSF (). After 4 d of G-CSF treatment, the majority (68 ± 1%) of 32D cells exhibited a polymorphonuclear appearance. Differentiated 32D cells bound to soluble E-selectin, and binding was abrogated by chelation of divalent cations or by prior treatment of the cells with sialidase. Protein extracts from the same cells were incubated with immunomagnetic beads coated with anti-CD44. Immobilized CD44 bound to soluble E-selectin in a manner similar to intact cells; binding was eliminated with EDTA or by sialidase treatment before lysis ( C). No binding was observed when beads were coated with an isotype-matched antibody binding αMβ2 integrin, or with control rat IgG (unpublished data). Immunoblot analyses revealed that CD44 was the sole protein purified from anti-CD44–coated beads (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20042014/DC1
). These results thus suggest that CD44 derived from mature myeloid cells interacts specifically with E-selectin. Further, we evaluated E-selectin binding specificity using CD44 extracted from a bone marrow stromal cell line (MS-5) and a brain endothelial cell line (bEnd.3), which express high levels of CD44 but do not bind to E-selectin. CD44 immunopurified from endothelial and stromal cells did not interact with E-selectin (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20042014/DC1
), underscoring differential posttranslational modifications of CD44 between hematopoietic and nonhematopoietic cells.
To investigate the contribution of O-linked and N-linked carbohydrates in the formation of ESLs on myeloid cells, we treated G-CSF–differentiated 32D cells with O
-sialoglycoprotein endopeptidase (OSGE) to remove O
-glycans. OSGE affected neither ESLs on the cell surface, nor E-selectin binding to immunopurified CD44 ( D), whereas P-selectin ligands were completely cleaved by OSGE (unpublished data). To assess the contribution of N-linked carbohydrates, ESLs were removed with sialidase and differentiated 32D cells were then cultured in the presence or absence of tunicamycin. De novo synthesis of ESL determinants on CD44 was dramatically inhibited (>90%, P = 0.002) by incubation with tunicamycin and the addition of OSGE to tunicamycin-treated cells did not further reduce E-selectin binding ( D). Tunicamycin treatment, however, did not affect P-selectin binding (which is dependent on O
-glycosylation; Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20042014/DC1
). Thus, CD44 derived from mature myeloid cell binds to E-selectin through N-linked, but not O-linked, glycans.
We next assessed E-selectin binding on primary mouse PMNs. BM and peripheral blood (PB) PMNs (Gr-1hi
) express high levels of CD44 but expression of ESLs is heterogeneous on BM PMNs whereas PB PMNs uniformly express high levels of ESLs ( E). E-Selectin binding to CD44 displayed a pattern similar to that of intact cells in that binding was lower on CD44 extracted from BM PMNs than on PB PMNs ( E), suggesting that the density of CD44 molecules that are appropriately decorated with E-selectin binding carbohydrates is greater on circulating PMNs than on maturing bone marrow PMNs. No binding was observed when beads were coated with anti-αM integrin or rat IgG (Fig. S2), or when CD44 was extracted from blood PMNs deficient in α(1,3) fucosyltransferase IV and VII (FucT IV/VII−/−
; E; reference 14
). These results further validate the binding specificity of the assay and indicate that CD44 is a physiological target of leukocyte FucTs. Fluid-phase binding of E-selectin to peripheral blood PMNs, an assay largely PSGL-1–dependent (10
), was significantly reduced in CD44−/−
PMNs compared with wild-type control PMNs (). Taken together, these data clearly indicate that CD44 from PB neutrophils binds to E-selectin.
E-selectin closely cooperates with P-selectin in promoting PMN–endothelial interactions and PMN extravasation; mice lacking both endothelial selectins have much more severe defects than either singly deficient animals (15
). E-Selectin–deficient mice display increased leukocyte rolling velocities, suggesting that it is critical in forming stronger adhesion bonds between endothelial cells and PMNs (19
). However, leukocyte rolling velocities were not altered in PSGL-1−/−
), suggesting that another leukocyte ESL mediates the slow rolling. We intercrossed CD44−/−
mice to characterize the function of CD44 in E-selectin–mediated leukocyte-endothelial interactions. Double knockout (DKO) animals were viable, fertile, and displayed a significant increase in circulating leukocytes, including PMNs, monocytes and eosinophils (Table S1, available at http://www.jem.org/cgi/content/full/jem.20042014/DC1
). We subjected age-matched male wild-type, CD44−/−
, and DKO mice to intravital microscopic examination of TNF-α–treated cremaster muscle to explore the ability of CD44 to interact with E-selectin in vivo. Consistent with previous studies (9
) and with the notion that P-selectin mediates most rolling activity in inflamed venules, the rolling fraction was significantly reduced in PSGL-1−/−
mice ( A and Table S2). Residual rolling activity in PSGL-1−/−
mice was further reduced by ~30% in mice lacking both PSGL-1 and CD44, although this did not reach the threshold statistical significance in a multigroup analysis of variance ( A). Although the average velocity of rolling leukocytes was similar between PSGL-1−/−
and wild-type mice ( and Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20042014/DC1
), leukocyte rolling velocities were significantly increased in CD44−/−
mice (P < 0.0001). Moreover, rolling velocities were further increased when both CD44 and PSGL-1 were absent ( B, P = 0.0002, comparison CD44−/−
with DKO group). In keeping with the velocity analyses, the median transit times of rolling leukocytes were much shorter in CD44−/−
than wild-type or PSGL-1−/−
mice, and even more rapid in DKO mice ( D). Because CD44 is known to interact with HA, we treated wild-type mice with hyaluronidase at a dose previously shown to alter CD44-mediated lymphocytes recruitment (11
). Hyaluronidase treatment neither influenced the leukocyte rolling flux nor affected leukocyte rolling velocities and transit times ( and Fig. S4). Thus, these data indicate that CD44 binding to E-selectin and not HA primarily controls the velocity of rolling, and that PSGL-1 can also contribute to this activity when CD44 is absent.
Figure 2. Intravital microscopy of TNF-α–stimulated cremaster muscle venules. Leukocyte behavior in cremasteric venules was recorded between 150 and 210 min after TNF-α administration for off-line analyses. (A) Rolling flux fraction in wild-type (more ...)
To investigate whether CD44 binding to E-selectin can mediate the extravasation of PMNs into inflammatory sites, we injected mice with thioglycollate, a chemical that induces a severe peritoneal inflammation. In this model, PMN recruitment is initially (0–4 h) P-selectin dependent but subsequently (8 h) requires E-selectin expression (15
). Previous studies have revealed no significant defect in PMN recruitment 8 h after thioglycollate injection in PSGL-1−/−
). Wild-type, CD44−/−
, and DKO mice were thus treated with thioglycollate, and the number of PMNs in the peritoneal exudate was evaluated 8 h after injection. Although there was no significant reduction in PMN counts in CD44−/−
mice, the recruitment of PMNs was significantly reduced (by 44%, P = 0.005) in DKO mice ( A). Because thioglycollate may not reproduce physiological inflammation, we also evaluated PMN extravasation elicited by the staphylococcal enterotoxin A (SEA) in a preformed air pouch model (20
). In preliminary experiments using P- and E-selectin–deficient mice, we ascertained that SEA-mediated PMN recruitment was selectin dependent (unpublished data). We then instilled SEA in dorsal skin pouches of mice from the four genotypes to assess the contribution of CD44 and PSGL-1 in this model. We found a severe reduction in the extravasation of PMNs 6 h after SEA injection in DKO mice compared with wild-type controls (77% reduction, P = 0.006), whereas the numbers of extravasated PMNs were not significantly altered in either singly-deficient mice ( B). To exclude further the possibility that the reduced recruitment of PMNs observed in DKO mice was due to CD44 binding to HA, we repeated the thioglycollate-induced extravasation experiments in PSGL-1−/−
mice treated with hyaluronidase (20 U i.v.) or vehicle. Hyaluronidase treatment did not significantly alter PMN recruitment ( C). Although we cannot completely rule out the possibility that extravascular HA may have remained available for PMN migration, the results from these two models strongly suggest that CD44 is an ESL that cooperates with PSGL-1 in PMN extravasation into inflamed sites.
Figure 3. Neutrophil extravasation into inflammatory sites. (A) Thioglycollate-induced peritonitis. Extravasated PMNs were determined 8h after the i.p. injection of thioglycollate (n = 7). (B) SEA-induced inflammation model. Extravasated PMNs were quantified 6 (more ...)
Because CD44 is expressed on both leukocyte and endothelial cells, we wished to clarify further whether CD44 deficiency on PMNs was sufficient to account for impaired PMN extravasation. We generated chimeric mice by transplantation of a mixture of PSGL-1−/−CD44+/+ and PSGL-1−/−CD44−/− BM-nucleated cells into lethally irradiated wild-type recipients. 6 wk after transplantation, >96% Gr-1hi leukocytes did not express PSGL-1. Peritoneal inflammation was then induced by thioglycollate for 8 h, and the ratios of PSGL-1−/−CD44+/+ PMNs over DKO PMNs in blood and peritoneal exudates were assessed by FACS ( A). In this competitive setting, we found that PSGL-1−/−CD44+/+ PMNs were preferentially recruited in the peritoneum (an approximate twofold increase) over those that did not express PSGL-1 and CD44 ( B), suggesting that neutrophil rather than endothelial CD44 plays a critical role for migration into inflammatory sites.
Figure 4. Competitive recruitment of neutrophils into inflammatory sites. Wild-type recipient mice were lethally irradiated and transplanted with mixture of BM cells from PSGL-1−/−CD44+/+ and PSGL-1−/−CD44−/− mice. (more ...)
Our data suggest that ESLs may have specialized functions. It is interesting to speculate that this may be controlled by the spatial distribution on the cell surface. For example, PSGL-1 is primarily a tethering molecule localized on the tip of microvilli (21
), whereas we show here that CD44, a receptor located on the cell body (22
), primarily controls rolling velocity. It is notable that β2 integrins, which can also mediate slow rolling (24
), are located on the cell body. Thus, our data are consistent with the notion that adhesion receptors located on microvilli may determine tethering efficiency but not rolling velocity (23
). As suggested by the partial defect in leukocyte rolling and recruitment of DKO mice, other functional ESLs exist on PMNs. The various ESLs may exert distinct functions in E-selectin–dependent adhesive and migratory activities. Further studies are needed to ascertain in vivo functions of major candidate ESL glycoproteins, including ESL-1 (expressed on mouse microvilli of myeloid cells; 7) and L-selectin (candidate ESL on human PMNs; 22
To investigate whether human neutrophil CD44 was a functional ESL, we purified peripheral blood PMNs from healthy donors and extracted PMN-derived human CD44 from cell lysates for the E-selectin binding assay. To control for binding specificity, we used sialidase-treated PMNs from the same donors and PMNs from a patient with leukocyte adhesion deficiency type II (LADII), characterized by a complete deficit in functional selectin ligands due to G588 nucleotide deletion in the GDP–fucose transporter gene (27
). As shown in A, healthy PMN-derived CD44 immobilized on beads bound to soluble E-selectin, and binding was abrogated when cells were treated with sialidase before the preparation of the lysates. In contrast, CD44 extracted from LADII PMNs did not appreciably bind to E-selectin ( B), despite normal expression of CD44 (, insets). Interestingly, a short incubation of LADII PMNs with recombinant FucTVI enabled CD44 to bind normally to E-selectin, indicating that CD44 on these cells possesses terminal N
-glycans that can be modified by extracellular FucT activity ( C). The rescue of selectin ligand activity by treatment with FucTVI may represent an effective therapy to prevent infectious episodes associated with this syndrome.
Figure 5. Human neutrophil CD44 binds to E-selectin. PMNs were purified from healthy donors or from a patient with LADII. PMNs were treated with sialidase or incubated with vehicle. (A) CD44 derived from healthy PMNs binds to E-selectin (empty red histogram), and (more ...)
In summary, these data clearly indicate that CD44 is a physiological ESL on mature human and mouse myeloid cells. CD44 is widely expressed in multiple cell types but only hematopoietic cells bind to E-selectin, indicating that the affinity for E-selectin is regulated by cell-specific posttranslational modifications. Selective binding to E-selectin (myeloid cells) or HA (activated T cells) endows CD44 with pivotal functions at the nexus of innate and adaptative immunity.