The mammalian lectin galectin-3 is a potent stimulus of human neutrophils, provided that the receptor(s) for the lectin has been mobilized to the cell surface before activation. We have recently shown that the receptors for galectin-3 are stored in intracellular mobilizable granules. Here we show supportive evidence for this in that DMSO-differentiated (neutrophil-like) HL-60 cells, which lack gelatinase and specific granules, are nonresponsive when exposed to galectin-3. Neutrophil granules were subsequently used for isolation of galectin-3 receptors by affinity chromatography. Proteins eluted from a galectin-3-Sepharose column by lactose were analyzed on SDS-polyacrylamide gels and showed two major bands of 100 and 160 kDa and a minor band of 120 kDa. By immunoblotting, these proteins were shown to correspond to CD66a (160 kDa), CD66b (100 kDa), and lysosome-associated membrane glycoprotein-1 and -2 (Lamp-1 and -2; 120 kDa). The unresponsive HL-60 cells lacked the CD66 Ags but contained the Lamps, implying that neutrophil CD66a and/or CD66b may be the functional galectin-3 receptors. This conclusion was supported by the subcellular localization of the CD66 proteins to the gelatinase and specific granules in resting neutrophils.
Galectin-3 is a mammalian lectin with affinity for β-galactoside-containing glycoconjugates and preferential binding to poly-N-acetyl-lactosaminoglycans (1). Several facts suggest that galectin-3 participates in inflammatory responses. It is produced by macrophages, and its level and rate of secretion are augmented by inflammatory mediators such as LPS and IFN-γ (2, 3). The extracellular galectin-3 may bind to other inflammatory cells and modulate or activate different cellular functions (4, 5). One such galectin-3-induced response is the superoxide anion production (oxidative burst) in neutrophil leukocytes (6, 7).
In a recent publication on galectin-3 interaction with human neutrophils we have shown that neutrophils isolated from peripheral blood are nonresponsive to galectin-3, while extravasated neutrophils produce superoxide in response to the lectin (7). During in vivo exudation, storage organelles, granules, are mobilized to the cell surface (7, 8), and we have suggested that a prerequisite for the galectin-3-induced neutrophil activation to occur is that receptors for galectin-3 be mobilized from these granules to the cell surface. By using different in vitro protocols for granule mobilization, the extent to which galectin-3 activates the neutrophil NADPH-oxidase was shown to parallel the mobilization of different subsets of granules to the cell surface. Galectin-3-binding receptor structures were thus shown to reside primarily in the gelatinase granules (and possibly also the specific granules) (7).
The promyelocyte cell-line HL-60 can be induced to differentiate into neutrophil-like cells (9). Such neutrophil-like HL-60 cells are frequently used as a model for studies of neutrophil function, because they contain many of the neutrophil receptors (e.g., the fMLP receptor and the complement receptor 3 (CR3)3) and have a functional NADPH-oxidase (10, 11). Differentiated HL-60 cells are, however, totally deficient in specific/gelatinase granules and in most of the proteins stored in these organelles in normal neutrophils (12, 13). Hence, if proteins in these granules were required for the induction of oxidative burst by galectin-3, then this response should also be lacking in HL-60 cells.
In this study we have investigated and characterized potential galectin-3 receptors in human neutrophils by exploiting differences/similarities between neutrophils and HL-60 cells.
Materials and Methods
Isolation of human neutrophils
Human neutrophils were isolated from buffy coats from healthy blood donors using dextran sedimentation and Ficoll-Paque gradient centrifugation (14). The cells were washed and resuspended (1 × 107/ml) in Krebs-Ringer phosphate buffer containing glucose (10 mM), Ca2+ (1 mM), and Mg2+ (1.5 mM; KRG; pH 7.3). The cell suspensions were stored on ice until use. This procedure allows for cells to be isolated with minimal granule mobilization (15).
Culture of HL-60 cells
The promyelocyte cell-line HL-60 (originally provided by Dr. R. C. Gallo, National Cancer Institute, Bethesda, MD) was passed once weekly in RPMI medium supplemented with 10% heat-inactivated FCS, penicillin (100 IU/ml), and streptomycin (100 μg/ml). The cells were induced to differentiate by addition of DMSO to a final concentration of 1.3% (9). The cells were harvested after 6 days, washed, resuspended in KRG (1 × 107/ml), and stored on ice until use. When activated by fMLP and PMA the differentiated cells responded with around 90 and 30% of the responses of peripheral blood cells, respectively (16, 17).
In vitro priming
To render the neutrophils sensitive to galectin-3 they were primed by pretreatment with the chemoattractant fMLP under conditions known to cause degranulation but not NADPH-oxidase activation (18). The cells were first incubated at 15°C for 5 min, after which fMLP (10−7 M final concentration) was added. The incubation was continued for 10 min at 15°C and then at 37°C for 15 min. The cells were washed twice in KRG to remove fMLP, resuspended in KRG (1 × 107 cells/ml), and kept on ice until use.
The mobilization of intracellular granules after in vitro priming was monitored by measuring the exposure of CR3 (CD11b/CD18) on the neutrophil surface using PE-conjugated monoclonal anti CD11b-Abs (M741, Dakopatts, Glostrup, Denmark; 10 μl for a cell pellet of 106 cells) and FACScan (Becton Dickinson, Mountain View, CA) (19).
Preparation of galectin-3 and galectin-3-Sepharose
Recombinant human galectin-3 (apparent Mr of 31 kDa) was produced in Escherichia coli and purified as previously described (20). The lectin was stored at 4°C in PBS (pH 7.2) containing lactose (150 mM). Before use in neutrophil activation, the lectin preparation was gel filtrated on a PD10 column (Pharmacia, Uppsala, Sweden) to remove lactose and then diluted to 400 μg/ml in KRG. Galectin-3-Sepharose was made using a method that involves oxidation of Sepharose and coupling of the protein by reductive amination (21).
Neutrophil NADPH-oxidase activity
The NADPH-oxidase activity was determined using a luminol/isoluminol-enhanced chemiluminescence (CL) system (22). The CL activity was measured in a six-channel Biolumat LB 9505 (Berthold, Wildbad, Germany), using disposable 4-ml polypropylene tubes with a 0.90-ml reaction mixture containing 106 neutrophils. The tubes were equilibrated in the Biolumat for 5 min at 37°C, after which the stimulus (0.1 ml) was added. The light emission was recorded continuously. To quantify intracellularly and extracellularly generated reactive oxygen species, respectively, two different reaction mixtures were used. Tubes used for measurement of extracellular release of superoxide anion contained neutrophils, HRP (a cell-impermeable peroxidase; 4 U), and isoluminol (a cell-impermeable CL substrate; 2 × 10−5 M) (23). Tubes used for measurement of intracellular generation of reactive oxygen species contained neutrophils, SOD (a cell-impermeable scavenger for O2−; 50 U), catalase (a cell-impermeable scavenger for H2O2; 2000 U), and luminol (a cell-permeable CL substrate; 2 × 10−5 M).
Subcellular fractionation was performed essentially according to the method described by Borregaard (24). In short, peripheral blood neutrophils isolated from buffy coats were treated with the serine protease inhibitor di-isopropylfluorophosphate (8 μM) and disintegrated by nitrogen cavitation (Parr Instruments, Moline, IL), and the postnuclear supernatant was centrifuged on a two-layer Percoll gradient (to isolate a mixture of specific and gelatinase granules), on a three-layer Percoll gradient (to isolate the specific and gelatinase granules separately) (25), or on a flotation gradient (to isolate the secretory vesicles from the plasma membranes (26). The gradients were collected in 1.5-ml fractions by aspiration from the bottom of the centrifuge tube, and the localization of subcellular organelles in the gradients was determined by marker analysis of the fractions. Vitamin B12 binding protein (marker for the specific granules) was determined using the cyanocobalamin technique as described by Gottlieb et al. (27). Gelatinase (marker for the specific and gelatinase granules) and myeloperoxidase (MPO) (marker for the azurophil granules) were measured using ELISA methods (28, 29). Alkaline phosphatase (marker for secretory vesicles and plasma membranes) was measured by hydrolysis of p-nitrophenyl phosphate (2 mg/ml) in the presence or the absence of Triton X-100 (0.4%) (30). The HLA class I Ag was determined by mixed ELISA as described by Bjerrum et al. (31).
Purification of galectin-3 binding proteins
The fraction containing the gelatinase and specific granules (β) was centrifuged at 100,000 × g for 90 min to remove the Percoll. The organelles were resuspended to 109 cell equivalents/ml in PBS, and an aliquot of the organelles corresponding to 2 × 108 cell equivalents was disrupted by freeze-thawing. The membranes were pelleted at 100,000 × g for 90 min, resuspended in 0.5 ml of 1% Triton X-100 in PBS, and incubated on ice for 30 min. The suspension was centrifuged at 100,000 × g to remove unsolubilized material. The supernatant was diluted 4-fold in PBS to give a 0.25% final concentration of Triton X-100. A galectin-3-Sepharose column of 2-ml bed volume was equilibrated with PBS-0.25% Triton X-100, after which the solubilized material was passed over the column. Unbound material was discarded, the column was washed with 20 vol of PBS-0.25% Triton X-100, and the bound proteins were eluted with 4 ml of 150 mM lactose in PBS-0.25% Triton X-100.
SDS-PAGE and Western blot
SDS-PAGE was performed according to the method of Laemmli (32) in 8% gels. Samples were diluted in nonreducing sample buffer, boiled for 5 min, and applied to the gels in volumes corresponding to the fractionated content of 5 × 106 cells. After electrophoresis the proteins were stained in the gel or transferred to polyvinylidene difluoride membranes using a Tris-glycine buffer system (33).
For detecting glycoproteins on the blotting membranes, the DIG Glycan Detection Kit (Boehringer Mannheim, Mannheim, Germany) was used as recommended. In short, carbohydrates were oxidized by periodate and labeled with digoxygenin. After addition of antidigoxygenin Abs labeled with alkaline phosphatase, carbohydrate-containing proteins were detected by addition of alkaline phosphatase substrate.
Membranes used for blotting with galectin-3 were blocked by incubation in PBS-Tween (0.05%, v/v, pH 7.3) containing gelatin (3%, w/v) for 1 h at room temperature before addition of galectin-3 (40 μg/ml) in PBS-Tween containing gelatin (1%, w/v) and incubation at room temperature for 1 h. The membranes were washed five times for 5 min each time in PBS-Tween and incubated with anti-galectin-3 Abs (anti-Mac-2 Abs; culture supernatant from the hybridoma M3/38; 1/25) in PBS-Tween containing gelatin (1%, w/v) for 1 h at room temperature. After washing twice, the membranes were incubated in HRP-labeled rabbit anti-rat Ig Abs (P0450, DAKO, Carpenteria, CA; 1/1000) for 1 h at room temperature and developed by adding peroxidase substrate (VIP Kit, Vector Laboratories, Burlingame, CA).
Membranes used for immunoblotting were first blocked in 1% BSA in PBS-Tween for 1 h and washed twice in PBS-Tween. Primary Abs were diluted as follows in blocking buffer: rabbit anti-human CEA (DAKO A0115; 1/500), mouse anti-human CD66a (a gift from Dr. Fritz Grunert, University of Freiburg, Freiburg, Germany; clone 4/3/17; 1/2,000), mouse anti-human CD66b (Serotec MCA216, clone 80H3; 1/10,000), and rabbit anti-human Lamp-1 and Lamp-2, respectively (gifts from Dr. Sven Carlsson, Umea University, Umea, Sweden; 1/1,000). The blots were incubated with primary Ab for 1 h and washed twice in PBS-Tween, after which HRP-labeled secondary Abs (rabbit anti-mouse Ig (DAKO P260, 1/2000) and goat anti-rabbit Ig (DAKO P0448), respectively) were added and the incubation was continued for 1 h. After extensive washing, the blots were developed by adding peroxidase substrate (VIP Kit, Vector Laboratories).
The fMLP, FITC, ATP, EGTA, p-nitrophenyl phosphate, PIPES, isoluminol, and luminol were obtained from Sigma (St. Louis, MO). SDS was obtained from Fluka Chemie (Buchs, Switzerland). Catalase, SOD, and HRP were purchased from Boehringer Mannheim. Dextran, Ficoll-Paque and Percoll were obtained from Pharmacia (Uppsala, Sweden). The m.w. standard proteins were purchased from Bio-Rad (Richmond, CA). [57Co]vitamin B12 was supplied by Amersham (Aylesbury, U.K.). Ionomycin was purchased from Calbiochem (La Jolla, CA). The Abs for the gelatinase-ELISA were gifts from Drs. Lars Kjeldsen and Niels Borregaard (Copenhagen, Denmark).
Neutrophils, but not HL-60 cells, respond to galectin-3
Neutrophils were in vitro primed with fMLP under the conditions previously described (7) to maximize priming while still keeping NADPH-oxidase activation by fMLP itself at a minimum. After removal of fMLP and addition of galectin-3, the primed neutrophils produced large amounts of superoxide both extracellularly and intracellularly, while the control (untreated) cells remained insensitive to galectin-3 (Fig. 1). No activity could be detected in the primed cells in the absence of stimulus (data not shown). In contrast, when differentiated HL-60 cells were pretreated with fMLP and then challenged with galectin-3, no extracellular superoxide was produced (Fig. 1, inset). Likewise, no intracellular response could be detected (data not shown), albeit this is a stimulus-independent effect, because HL-60 cells lack specific granules and consequently cannot assemble a functional NADPH-oxidase at an intracellular site (16). The inability to respond to galectin-3 was seen also in unprimed as well as undifferentiated HL-60 cells (data not shown).
Neutrophils, but not HL-60 cells, express increased numbers of CR3 and galectin-3 receptors on their surface after priming
The cell surface expression in primed (fMLP-treated) and unprimed (control) cells of CR3 (Fig. 2, top), a marker for the specific and gelatinase granules, and galectin-3 binding proteins (Fig. 2, bottom) was compared using fluorescent reagents and flow cytometry. For neutrophils, the number of CR3 and galectin-3 binding proteins both increased after in vitro priming. For HL-60 cells the level of both markers were lower by an order of magnitude, and no increase was observed after priming.
Taken together, these activation and binding studies support our previous finding that mobilization of gelatinase/specific granules and increased expression of galectin-3 binding proteins on the neutrophil surface is a prerequisite for neutrophil responsiveness to galectin-3 (7). Therefore, the gelatinase/specific granules were used as source for isolation of galectin-3 receptors.
CD66 and Lamps are major galectin-3 binding proteins in gelatinase and specific granules
Galectin-3 binding proteins were isolated from carefully fractionated gelatinase/specific granules (Fig. 3,A) by affinity chromatography on galectin-3-Sepharose. The specifically bound proteins were eluted with lactose, a competitive inhibitor of galectin-3, and analyzed by SDS-PAGE followed by Western blotting and probing with specific Abs. Silver-stained SDS-PAGE indicated two major galectin-3 binding proteins of Mr 100 and 160 kDa and a minor band of 120 kDa (Fig. 4). The Mr, vesicular localization (26, 34, 35), and galectin-3 binding (36, 37, 38) agreed with previous reports for proteins of the CD66 family (CD66b and CD66a, 100 and 160 kDa, respectively) and Lamp-1 and -2 (∼120 kDa). Probing with anti-CD66 and anti-Lamp Abs confirmed the presence of these proteins as components among the galectin-3 binding proteins (Fig. 4). Specific labeling for carbohydrate (Fig. 4) showed that they were heavily glycosylated, as also indicated by the relatively diffuse bands.
DMSO-differentiated HL-60 cells express Lamp-1 and –2, but not CD66a or CD66b
To address the functional role of CD66a, CD66b, and the Lamps as galectin-3 receptors we compared neutrophils with differentiated HL-60 cells. Probing whole cell extracts with galectin-3 followed by anti-galectin-3 Abs revealed two major bands at Mr 100 kDa and 160 kDa in neutrophils, corresponding by m.w. to CD66a and CD66b, while no binding was seen in the HL-60 cells (Fig. 5).
Probing of Western blots of the neutrophil and HL-60 cell extracts with anti-Lamp Abs revealed about equal amounts of Lamps in both cell types (Fig. 6) in agreement with previous studies (39). However, probing with anti-CD66a and anti-CD66b Abs revealed the presence of these proteins in neutrophils, but barely (CD66a) or not at all (CD66b) in HL-60 cells (Fig. 6).
The subcellular localization of CD66 is consistent with the proposed galectin-3 receptor distribution
Neutrophils were fractionated on a three-step Percoll gradient to separate gelatinase granules from specific granules (Fig. 3,B) and on a flotation gradient to separate secretory vesicles from plasma membranes (Fig. 3,C). When probing proteins from the different granules/vesicles with specific Abs for CD66a and CD66b, these proteins were found mainly in the gelatinase and specific granules and much less in the secretory vesicles and plasma membrane of unprimed cells (Fig. 7). In contrast, Lamp-1 and -2, although present in the fractions containing specific and gelatinase granules, were even more prominent in the secretory vesicle fractions and were also present in the fractions enriched in plasma membrane (Fig. 7).
The basis for this study was our previous finding that galectin-3 is a new inflammatory mediator that activates human neutrophils that are in a primed state (7). We could also show that the functional alteration in the neutrophils from being nonresponsive (in peripheral blood) into being galectin-3 responding (e.g., in an inflammatory exudate) strongly correlates with receptor mobilization following translocation of granules to the cell surface. The galectin-3 receptor(s) was shown to be a component of the gelatinase/specific granules, but its identity was not established. Here, we have defined CD66a and CD66b as galectin-3 receptor candidates responsible for inducing neutrophil NADPH-oxidase activation.
In other cell types, both soluble and membrane-bound galectin-3-binding molecules have been isolated and characterized, but very few studies have been able to verify the involvement of one specific molecule in cell activation by galectin-3. In the T lymphoblastoid Jurkat cells, galectin-3 binds CD98 (4F2 Ag), triggering a Ca2+ influx (40). This galectin-3 binding protein is also present in macrophages and has been isolated from these cells on a galectin-3 affinity column together with the α subunit (CD11b) of CR3 (CD11b/CD18, Mac-1), Lamp-1, and Lamp-2 (36). The human melanoma cell line A375 also exposes galectin-3-binding Lamp-1 and -2 (37), and circumstantial evidence indicates that these molecules are responsible for galectin-3 binding to such cells (41). In addition, A375 cells secrete soluble galectin-3-binding glycoproteins of 70 and 98 kDa, the latter corresponding to the Mac-2 binding protein (Mac-2-BP; human lung tumor L3 Ag) (37, 42). In colon carcinoma cells, coimmunoprecipitation and affinity chromatography on immobilized galectin-3 identified several galectin-3-interacting proteins, including CD66 and the Lamps (38).
In human neutrophils, we here identify four galectin-3-binding proteins isolated by affinity chromatography: CD66a, CD66b, Lamp-1, and Lamp-2, with CD66a and CD66b being the most prominent. Our suggestion that CD66a and CD66b are the most likely receptor candidates is based on the findings 1) that differentiated HL-60 cells that are nonresponding to galectin-3 contain Lamp-1 and Lamp-2, but no CD66a or CD66b, while galectin-3-responding neutrophils contain all four molecules; and 2) that the subcellular localization of CD66a and CD66b, but not of the Lamps, is in agreement with the granule mobilization needed for the cells to respond to galectin-3.
In resting neutrophils CD66a and CD66b have been suggested to localize to the specific granules (34, 35). Recently, the specific granules have been shown to comprise at least two different granule subsets, the classical specific granules that contain vitamin B12 binding protein and lactoferrin and the gelatinase granules that are deficient in these two proteins but abundant in gelatinase. Here we show that CD66a and CD66b are present in both granule types in comparable amounts. This is consistent with their role as galectin-3 receptors as in our previous study the mobilization of gelatinase granules and specific granules most closely correlated with acquisition of responsiveness to galectin-3 (7). The small amounts of CD66a and CD66b found in the secretory vesicles may explain the appearance of CD66a at the cell surface after short term activation with fMLP of freshly isolated neutrophils (which normally do not express cell surface CD66) (43). Such mild treatment does not result in mobilization of either gelatinase or specific granules, whereas the secretory vesicles are readily mobilized (our unpublished observation).
Lamp-1 and Lamp-2 are regarded as classical lysosomal membrane proteins. In neutrophils, however, the Lamps were found to be absent from isolated azurophil granules, until recently regarded as a lysosome equivalents, but were instead present in organelles cofractionating with the specific granules, gelatinase granules, and secretory vesicles (26). A recent immunohistochemical study indicates that the Lamps are not true components of the granules/vesicles, but are instead localized in multilaminar compartments and multivesicular bodies (44), in analogy to HL-60 cells, which store the Lamps in multivesicular bodies (39). The subcellular localization to these nonsecretory organelles could thus explain the difficulty of mobilizing the Lamps to the cell surface in neutrophils, while they much more readily go to the phagosome (26, 44). Taken together, the presence of the Lamps in the nonresponding (with regard to galectin-3) HL-60 cells in combination with the subcellular localization to nonsecretable organelles indicates that these molecules, although galectin-3 binding, are not functional galectin-3 receptors in neutrophils.
A role for CD66a and CD66b as galectin-3 receptors is made further plausible by the fact that these molecules can induce intracellular signals leading to oxidative burst upon ligation and cross-linking. For example, ligation of CD66a with single-chain Fv fragments in prestimulated neutrophils (in which CD66a is exposed on the cell surface) induces NADPH-oxidase activity (43). Cross-linking of Abs binding to CD66b in neutrophils also results in the production of oxygen radicals (45) as well as in increased adhesion to endothelial cells (46). However, in contrast to CD66a, CD66b, which is linked to the membrane via glycosyl phosphatidylinositol, probably cannot transduce signals into the cell by itself, but may require coligation with CD66a (47). Although little is known about the specific signal transduction events induced by CD66a and CD66b, tyrosine phosphorylation appears to be one important component (48, 49). The inhibition of galectin-3 activation by the tyrosine kinase inhibitor genistein (our unpublished observation) is thus consistent with signaling through CD66a and CD66b.
When primed neutrophils were preincubated with anti-CD66a or anti-CD66b Abs before stimulation with galectin-3, no inhibitory effect of the Abs could be detected on either of the NADPH-oxidase responses (data not shown). However, we have no knowledge about the epitopes engaged by the Abs and cannot exclude that galectin-3 may bind a different epitope and thereby induce activity regardless of binding of Ab to the glycoproteins.
Because signaling through CD66a and CD66b clearly differs, it is possible that galectin-3 activates two different signaling pathways. This gains support from the fact that two different NADPH-oxidase responses are induced, extracellular and intracellular, respectively, which are executed by two different NADPH-oxidase pools. We have strong indications that these two pools can be activated by separate signal transduction pathways.4 The possibility that the two pathways are launched by two different receptor molecules is intriguing, but requires much more extensive investigation to be verified.
The excellent technical assistance of Marie Samuelsson is greatly appreciated.
This work was supported by the Fredrik and Ingrid Thuring Foundation, the Swedish Medical Research Council, the Swedish Society of Medicine, the Anna-Greta Crafoord Foundation for Rheumatological Research, the King Gustaf V 80-Year Foundation, the Lars Hierta Foundation, and the Magnus Bergvall Foundation.
Abbreviations used in this paper: CR3, complement receptor 3; CL, chemiluminescence; SOD, superoxide dismutase.
A. Karlsson, J. B. Nixon, and L. C. McPhail. 1999. Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways; dependent or independent of phosphatidylinositol 3-kinase. Submitted for publication.