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a Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany
b Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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Key Words: adhesion • tumor cells • metastasis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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Although the selectins have been shown to interact weakly with sialylated, fucosylated lactoseaminoglycans such as sialyl-Lewisx (sLex) and other sialylated moieties that are widely distributed on the surface of leukocytes (12), they bind with much higher affinity to glycans displayed on a limited number of glycoproteins (5, 13). One high-affinity selectin ligand is P-selectin glycoprotein ligand-1 (PSGL-1)2 (14). PSGL-1 is a sialylated, mucin-like disulfide-linked homodimer that displays mainly O-linked glycans and at least one sulfated tyrosine residue at the N-terminus of the molecule (15–18). PSGL-1 is expressed on most leukocytes, where it appears to account for most of the high affinity binding to P-selectin and is essential for neutrophils to roll on P-selectin at physiological shear (19, 20).
We have previously identified and characterized another ligand for P-selectin. Human CD24 on a breast (KS) and a small cell lung carcinoma (SW-2) cell line, both negative for PSGL-1, can bind to P-selectin under static conditions (21). Latex beads coated with CD24 purified from these two carcinoma cell lines or from neutrophils bind specifically to immobilized P-selectin-IgG. The carcinoma cells and CD24-coated beads derived from these cells bind to activated platelets and P-selectin transfected Chinese hamster ovary cells in a P-selectin dependent manner, and this binding is blocked by soluble CD24 (21).
CD24 is a mucin-like glycosylphosphatidylinositol-linked cell surface glycoprotein. It consists of a small protein core comprising only 27 amino acids which is extensively glycosylated (22). Nearly half of the amino acids in CD24 are serine and threonine residues that are potential sites for O-linked glycosylation. CD24 is expressed at the early stages of B-cell development. It is highly expressed on neutrophils and many human carcinomas, but is absent on T-cells, monocytes or normal adult tissues (23–25).
We hypothesized that CD24 would also mediate cell capture and rolling on P-selectin and therefore examined whether CD24 positive tumor cells could adhere to P-selectin under flow conditions in a parallel plate flow chamber. Our results indicate that CD24 can function as a physiological counter receptor for P-selectin and mediate rolling of the breast carcinoma cell line KS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREFERENCES |
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Cells
The human breast carcinoma cell line KS (29) was provided by B. Gückel (University of Heidelberg, Germany). The breast carcinoma cell lines KS, McF-7 (30) and SkBr3 (31) and the small cell lung cancer cell line H69 (32) were cultivated in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), at 37°C, 5% CO2 and 100% humidity. The human promyelocytic cell line, HL-60, and the human lymphoma cell line, SkW3, were maintained in RPMI 1640 with 10% FCS. The murine pre-B lymphocytic cell line, 300.19, was maintained in RPMI 1640 supplemented with 10% FCS and 10 µM 2-mercaptoethanol. The cell line 300.19 stably transfected with cDNA encoding human L-selectin or P-selectin (33) were cultured under the same conditions. All cell culture media contained glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 g/ml) (Gibco BRL, Grand Island, N.Y.).
Laminar flow assay
Human P-selectin-IgG chimera was diluted to a concentration of 4 µg/ml in tris-saline-azide (TSA), pH 9.5 (20 mM Tris, 140 mM NaCl, 0.025% azide) and adsorbed to polystyrene slides cut from bacteriological petri dishes (Falcon 1058). The slides were blocked with 3% human serum albumin and fitted into a parallel plate laminar flow chamber (34), which was mounted on the stage of an inverted phase contrast microscope (Diaphot-TMD, Nikon, Garden City, N.Y.). Adhesive interactions between cellular Fc receptors and the Fc domain of P-selectin-IgG were eliminated by preincubation of the adsorbed selectin substrate with a polyclonal F(ab')2 (10 µg/ml) against human IgG Fc. For antibody inhibition studies, the adsorbed selectin substrate or cell suspensions were incubated with antibodies (10 µg/ml) for 20 min before initiation of flow. For continuous flow assays, the cells were resuspended at a concentration of 0.5–1 x 106/ml in HBSS supplemented with 10 mM HEPES and 2 mM Ca2+ and drawn through the flow chamber at room temperature using a syringe pump (Havard Apparatus, South Natick, Mass.). The number of bound cells was quantified from videotape recordings of 10–20 fields of view obtained while scanning the lower plate of the flow chamber using a 10x objective. For rolling velocity analysis, video images were captured using NIH Image v.1.57, and the displacement of individual leukocytes under shear conditions was tracked at 1–5 s intervals. Critical velocity was defined as the velocity of a non-interacting cell in a shear flow near the wall of the flow chamber (35). Cells with a translational velocity lower than the critical velocity were defined as rolling.
Affinity purification of CD24
CD24 was purified by affinity chromatography on a mAb SWA11 column as described previously (36). Briefly, KS cells were lysed in the presence of Nonidet P-40 and the lysates were passed over the antibody column. The column was eluted with 100 mM diethylamine, pH 11.5, containing 50 mM-octylglucoside as detergent. Eluted antigen was neutralized, dialyzed against PBS pH 7.4, and stored at -70°C. CD24-coated beads were prepared by incubating purified CD24 (150 µg/mL) with polystyrene beads (Serva, Heidelberg, Germany). Remaining binding sites were blocked by incubation with 1% BSA in PBS for 1 h. CD24 expression on beads was quantified by flow cytometry.
Enzyme treatments
Cells (5 x 106/ml) were incubated in 500 µL DMEM medium without FCS in the presence of 500 mU PI-PLC (Boehringer, Mannheim, Germany) for 120 min at 37°C. Cells were then washed and analyzed for expression of CD24 by flow cytometry or infused into the flow chamber for adhesion assays.
Flow cytometry
Cells or antigen coated beads were stained by indirect immunofluorescence as described (36). Stained cells or beads were analyzed with a FACScan fluorescence activated cell analyzer (Becton Dickinson, San Jose, Calif.) using the indicated primary mAbs followed by either FITC- or phycoerythrin-conjugated goat anti-mouse antibodies. For cell sorting, the KS cells were stained initially with the CD24 mAb ML-5, followed by FITC conjugated goat anti-mouse IgG. Cells with low and high levels of CD24 expression were sorted on a FACS Vantage sorter (Becton Dickinson, San Jose, Calif.). Sorted cells were used immediately in adhesion assays.
Intravital microscopy
C57BL/6 or E-selectin-deficient mice (provided by A. L. Beaudet, Baylor College of Medicine, Houston, Tex.) were anesthetized with an intraperitoneal injection of ketamine hydrochloride (100 mg/kg, Ketalar, Parke-Davis, Morris Plains, N.J.) after pretreatment with xylazine and atropine (0.1 mg/kg ip, Elkins-Sinn, Cherry Hill, N.J.). Mice were pretreated 2 h before surgery with an intrascrotal injection of 0.5 µg murine TNF-
(Genzyme, Cambridge, Mass.) in 0.3 ml isotonic saline. A fluid-filled, heparinized catheter was placed into the proximal part of the right femoral artery and advanced toward the branching section of the internal iliac artery from the common iliac artery. The ipsilateral cremaster muscle to the femoral catheter was exteriorized and superfused with bicarbonate-buffered saline at 37°C. KS cells were labeled with 0.5 µg/mL calcein AM (Molecular Probes, Eugene, Ore.) for 30 min at 37°C. Cells (5–10x106/mL) were injected as a 0.1 mL bolus into the cremaster microcirculation via the femoral artery catheter. Microscopic observations were made on a intravital microscope (Zeiss Axioskop, Thornwood, N.Y.) with a saline immersion objective (SW 40/0.75 numerical aperture). Larger venules with diameters between 52 and 62 µm were observed and video recordings were made through a CCD camera system (model VE-1000CD, Dage-MTI, Michigan City, Ind.) on a Panasonic S-VHS recorder. Measurements of KS cell rolling were performed using stroboscopic epifluorescence illumination (60 sec-1, Strobex; Chadwick Helmuth, Mountain View, Calif.). For each introduction of cells, video scenes of approximately 10 min duration were recorded (250–300 passing cells). The centerline velocities of the respective vessels were calculated using the advance of free-flowing labeled cells in the center of the vessels in consecutive video frames as described (37). After initial injection of cells, flow was transiently reduced due to lodging of cells in capillaries. However, in all experiments flow returned to baseline values within 45 sec. Shear rates were determined as 
w = 2.12 x 8Vb/d. Where w is the wall shear rate, Vb is the mean blood flow velocity, d is the diameter of the vessel, and 2.12 is a median empirical correction factor obtained from actual velocity profiles measured in microvessels in vivo (38). For each venule, a critical velocity was determined as the minimal velocity of a freely flowing KS cell traveling close to the vessel without adhesive interactions (37).
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RESULTS |
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Fig. 1).
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Figure 2A
shows that immobilized human P-selectin-IgG supported rolling of HL-60 and SkW3 cells as expected, but also supported rolling of KS cells under flow conditions. Attachment of KS cells under flow was shear stress dependent. KS cells were allowed to attach at low shear stress (0.25 dyn/cm2) and shear stress was increased at 0.25 dyn/cm2 increments. KS cell rolling gradually decreased with increasing shear stress and was completely eliminated at 2.75 dyn/cm2 ( Fig. 2A). In contrast, HL-60 cells, as well as SkW3 cells, continued to maintain adhesive interactions and roll under the same conditions. Attachment and rolling of KS, HL-60 and SkW3 cells were P-selectin and Ca2+-dependent, as demonstrated by complete inhibition of rolling after treatment of the P-selectin-IgG substrate with the P-selectin mAb G1, or after addition of 5 mM EDTA to the perfusion media ( Fig. 2B). The PSGL-1-negative nature of KS cells was further confirmed by the inability of the PSGL-1 mAb KPL-1 to block KS cell rolling on P-selectin, whereas rolling of HL-60 cells on P-selectin was completely inhibited by mAb KPL-1 treatment (data not shown).
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Rolling velocity analysis of KS cells in comparison to HL-60 and SkW3 cells
KS, HL-60 and SkW3 cells formed stable rolling interactions with immobilized P-selectin and rolled at velocities below the critical velocity. The critical velocity was experimentally determined at 1 dyn/cm2 for nonadherent cells traveling adjacent to the lower wall of the flow chamber (796±155 m/s for KS cells, 759±128 m/s for HL-60 cells and 834±104 m/s for SkW3 cells). Displacement tracings for individual HL-60 and SkW3 cells rolling on P-selectin-IgG at 1 dyn/cm2 were comparable, whereas KS cells traveled a much greater distance in the same period of time (data not shown). Rolling velocity histograms demonstrated that KS cells rolled on P-selectin-IgG at higher velocities than HL-60 and SkW3 cells (range of 10–35 µm/s for 95% of rolling KS cells vs. a range of 1–7 µm/s for 95% of rolling HL-60 and 4–8 µm/s for 95% of SkW3 cells) ( Fig. 3).
These findings indicate that KS cells express a ligand for P-selectin, distinct from PSGL-1, that supports rolling under flow conditions. This P-selectin ligand activity appears to be less efficient at mediating rolling and more shear stress sensitive than PSGL-1.
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Effect of PI-PLC treatment on P-selectin ligand activity
To determine whether CD24 is required for rolling of KS cells under flow conditions, KS cells were treated with PI-PLC before infusion through the flow chamber. Phosphatidylinositol-phospholipase C (PI-PLC) (39) specifically cleaves GPI-anchored molecules, such as CD24. The cleavage of CD24 on KS and HL-60 cells was confirmed by flow cytometry. Compared to mock-treated KS cells, treatment of KS cells with PI-PLC resulted in inhibition (68%) of attachment and rolling on immobilized P-selectin-IgG under flow conditions ( Fig. 4
A and C). By contrast, PI-PLC treatment had no effect on HL-60 cell rolling ( Fig. 4B). In addition, we observed a change in the rolling behavior at the single cell level. PI-PLC treatment of KS cells disrupted continuous rolling ( Fig. 5A)
resulting in an irregular and uneven rolling pattern as shown by variation in the single cell velocity as a function of time ( Fig. 5B). Furthermore, the enzyme treatment caused a clear increase in the rolling velocities of the cells that continued to roll (from an average of 27 µm/s to 54 µm/s at a shear rate of 1 dyn/cm2, data not shown). Some cells also formed only brief adhesive interactions after which they skipped to velocities above the critical velocity, which resulted in cell detachment.
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Treatment of KS cells with soluble CD24 blocks rolling
Since function-blocking antibodies to human CD24 have not been described (21), we used soluble CD24 (sCD24) to further assess the specificity of CD24 mediated rolling on P-selectin. sCD24 isolated from KS cells was able to reduce the accumulation of KS cells on P-selectin-IgG at 1 dyn/cm2 from 22 to 9.8 cells/mm2. By contrast, sCD24 isolated from mouse ESb cells, which does not bind to P-selectin (36), did not inhibit KS adhesion to P-selectin-IgG (data not shown). In addition, sCD24 derived from KS cells failed to block binding of HL-60 cells or SkW3 cells to P-selectin-IgG under flow conditions. These data further support the involvement of CD24 in mediating binding of a PSGL-1-negative carcinoma cell line to P-selectin.
KS cell rolling on P-selectin is dependent on CD24 expression level
KS cells with high and low expression of CD24 were used to directly correlate CD24 expression with rolling on P-selectin. For this purpose KS cells were sorted by flow cytometry in two populations, either high (CD24high) or low (CD24low) expression levels of CD24. As shown in Fig. 6,
there was a clear correlation between attachment rate on P-selectin and expression level. CD24low KS cells showed a decrease in attachment, whereas CD24high cells adhered avidly to P-selectin-IgG under flow conditions. Together, our data establish that CD24 is necessary for rolling of KS cells on P-selectin.
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Glycosylation requirement for rolling on P-selectin
Previous data from our group (21) have shown that CD24-positive tumor lines, which do not express detectable levels of PSGL-1, were able to bind to P-selectin under static conditions. From this panel we selected three additional tumor lines (two breast carcinoma cell lines, McF-7 and SkBr3, and one small cell lung cancer cell line, H69) and investigated their interaction with P-selectin-IgG under flow conditions. In contrast to KS cells, none of these other cell lines was able to roll on P-selectin under flow conditions (data not shown).
Earlier studies have shown that a wide variety of tumor cells are able to bind to E- and/or P-selectin (7, 9, 40–42) and that in some of these tumor lines (43) the ability to bind selectins correlates with the expression of sLex. We have found that KS cells also attach and roll on E-selectin-IgG under flow conditions (1 dyn/cm2), but treatment with PI-PLC did not inhibit KS cell adhesion to E-selectin (data not shown). This finding suggests that CD24 is not required for KS cell binding to E-selectin under flow conditions and that recognition of E-selectin may be mediated by other glycoproteins decorated with sLex epitopes. To ascertain the role of sLex in KS binding to P-selectin, we stained tumor cell lines with the sLex mAb AM-3. KS cells, which attached specifically to P-selectin under flow, stained with mAb AM-3, whereas the cell lines McF-7, SkBr3, and H69, which failed to bind P-selectin, reacted only weakly or not at all with mAb AM-3.
To address whether CD24 is decorated with sLex, we purified CD24 from KS and McF-7 cell lines and coated the material on beads. Beads coated with CD24 derived from KS cells stained positively with both CD24 mAb ML5 and sLex mAb AM-3. In contrast, beads coated with CD24 purified from McF-7 cells stained only for CD24 ( Fig. 7).
These findings suggest that adhesive interactions under flow between P-selectin and CD24 require the enzymatic machinery necessary to decorate the cell surface with the sLex determinant. It is important to note that expression of sLex was not able to mediate effective carcinoma cell rolling on P-selectin without sufficient expression of CD24 (
Fig. 6).
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P-selectin transfectants roll on sCD24 affinity purified from sLex positive cells
To investigate whether CD24 is sufficient to mediate attachment and rolling, we incorporated sCD24, which was purified to homogeneity from KS cells (21), as a substrate in the flow chamber and perfused 300.19 cells stably transfected with either P-selectin (300.19P) or L-selectin (300.19L). As shown in Fig. 8,
300.19P transfectants attached and rolled on immobilized sCD24 under flow conditions, whereas 300.19L transfectants did not. Attachment and rolling of 300.19P on immobilized sCD24 was completely blocked by incubation of the transfectants with an antibody against P-selectin (mAb G1). These data indicate that CD24 derived from KS cells is sufficient to support P-selectin dependent rolling.
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P-selectin-dependent KS cell rolling on vascular endothelium in vivo
To determine whether CD24 can mediate P-selectin-dependent rolling on vascular endothelium, we injected KS cells into the femoral artery of TNF-treated mice and observed cell transit through postcapillary venules of the exteriorized cremaster muscle. In wild-type mice, approximately 25% of the KS cells passing through the observed vessel rolled along the vessel wall. This fraction was sharply reduced after injection of the P-selectin mAb RB40.34 (44) (Table I). KS cell rolling was similar in postcapillary venules of E-selectin-deficient mice (Table I). These data show that CD24 can mediate KS cell rolling on inflamed endothelium in vivo.
DISCUSSION
CD24 was previously identified as potential ligand for P-selectin, because CD24 supports the binding of human tumor cells to platelets and P-selectin transfectants in a static cell adhesion assay (21). However, structures may interact with selectins under static conditions without serving as specific ligands under physiological flow conditions (45). To date, only PSGL-1 has been shown to function as a ligand for P-selectin in the presence of shear stress (19).
The parallel plate flow chamber assay is a high stringency cell adhesion assay that mimics physiologic flow conditions. Thus, we used this assay system to ascertain the physiological role of CD24 as a ligand for P-selectin. Our data show that CD24 on the breast carcinoma cell line, KS, was required for mediating rolling on P-selectin, because low expression levels or cleavage of CD24 resulted in inhibition of attachment and rolling. In addition, cleavage of CD24 changed the rolling behavior of single cells from continuous to irregular, skipping-like rolling. Furthermore, purified CD24 specifically supported rolling of P-selectin transfectants. Finally, we demonstrated P-selectin-dependent KS rolling on vascular endothelium of postcapillary venules in vivo.
Blocking CD24 function with excess soluble CD24 or by removing CD24 from the cell surface with PI-PLC significantly reduced attachment and rolling of KS cells, but had no effect on HL-60 cells, which express PSGL-1 in addition to CD24. Apparently, PSGL-1 dominates the rolling behavior on P-selectin in cells that express both CD24 and PSGL-1. This is supported by the observation that mAbs to PSGL-1, PL-1 (19) or KPL-1 (28), completely block HL-60 cell rolling on purified or recombinant P-selectin, even though CD24 is still present. Of note, the expression level of CD24 on HL-60 cells is 5–10 times lower than that on KS cells ( Fig. 1). At this level of expression, CD24 may be incapable of mediating significant rolling under flow.
A large body of evidence indicates that sialylated and fucosylated cell surface determinants like sLex play a crucial role in adhesion by selectins (2, 5). Earlier studies have shown that in some tumor lines the ability to bind to selectins correlates with the expression of sLex (7, 9, 40–42). We therefore tested our panel of cell lines by flow cytometry and Western blot analysis for sLex expression and found sLex, as detected by mAb AM-3 (27), only on KS cells, but not on McF-7, SkBr3 or H69 cells. These findings suggest an important role for sLex in mediating CD24 dependent rolling, whereas sLex appears not to be critical for mediating binding of CD24 to P-selectin under static conditions (21). In the biosynthesis of sialylated, fucosylated glycoproteins, the addition of fucose is thought to be an important regulatory step for the expression of selectin ligand activity (2). Fucosylation requires specific fucosyltransferases (Fuc-TVII and Fuc-TIV) (46, 47), which are expressed by leukocytes. The expression of sLex is considered to be a marker for the presence of the enzymatic machinery is required for the construction of biologically relevant selectin ligands. Our data show that expression of CD24, as well as its modification by sLex, are required for PSGL-1 independent rolling of KS cells on P-selectin. It appears that the posttranslational modifications required by CD24 for function are similar to those required by PSGL-1. The role of sLex-decorated CD24 on tumor cells may be twofold, both of them potentially relevant for metastasis. On one hand, tumor cells metastasizing through a hematogenic pathway are known to bind platelets while traveling in the bloodstream (48–50). Since activated platelets express P-selectin, CD24 may be a physiological ligand important for P-selectin binding. Platelet binding to metastasizing tumor cells is likely to occur at the point at which the primary tumor invades the vascular system, exposing thrombogenic tumor cells to the blood. Activated platelets expressing P-selectin may bind to CD24-positive tumor cells protruding into the blood vessel, a process that requires adhesion under shear stress. Although platelet rolling on endothelial cells has been described (51), platelet rolling on tumor cells has not been observed directly. However, in vitro studies have established the ability of tumor cells to roll on endothelial cells (42, 52) and platelets (8). Additional platelet binding to tumor cells may occur while the tumor cells are in transit in the bloodstream.
Tumor cell metastasis is known to be facilitated by inflammation (53, 54). Under inflammatory conditions, endothelial cells express P-selectin (4, 6) and other adhesion molecules on their surface. Several studies suggest that tumor cells arriving in the target organ may roll on activated endothelium before being able to arrest and proliferate (52, 53). Our data show that CD24 expressed on tumor cells can support rolling on P-selectin. Hence, the CD24-P-selectin pathway may be an important element in recruiting tumor cells to target organs. There is precedent for cell surface adhesion molecules being important for tumor cell metastasis, including E-selectin and its ligands in colon carcinoma (10, 41, 42, 55), VLA-4 and VCAM-1 in melanoma (56, 57), b integrins in breast carcinoma (58), and L-selectin in lymphomas (59). Our data suggest that CD24 binding to P-selectin may be an additional adhesion pathway for tumor cell metastasis. This is further supported by studies showing a correlation between CD24 expression and cancer metastasis (23, 60–62). More direct studies addressing the metastatic mechanisms of CD24-bearing tumor cells will be required to fully understand the role of the CD24-P-selectin interaction in tumor pathophysiology.
In summary, our data demonstrate that CD24 expressed by the breast carcinoma cell line KS is necessary and sufficient to support P-selectin dependent rolling in vitro and in vivo. This finding establishes CD24 as a second glycoprotein ligand for P-selectin, which appears to be functionally important on cells that do not express PSGL-1.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: Fuc-TIV, fucosyltransferase IV; Fuc-TVII, fucosyltransferase VII; GPI, glycosylphosphatidylinositol; HBSS, Hanks' balanced salt solution; PI-PLC, phosphatidylinositol-phospholipase C; PSGL-1, P-selectin-glycoprotein ligand-1; sLex, sialyl-Lewisx; TSA, Tris-saline-azide; 300.19L, murine pre-B lymphocytic cell line 300.19 transfected with L-selectin; 300.19P, 300.19 cells transfected with P-selectin.
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REFERENCES |
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