Biological Activities of Ecalectin: A Novel Eosinophil-Activating Factor1 Ryoji Matsumoto,2* Mitsuomi Hirashima,† Hirohito Kita,* and Gerald J. Gleich* Ecalectin, produced by Ag-stimulated T lymphocytes, is a potent eosinophil-specific chemoattractant in vitro as well as in vivo and thus is implicated in allergic responses. Ecalectin differs structurally from other known eosinophil chemoattractants (ECAs); ecalectin belongs to the galectin family defined by their affinity for -galactosides and by their conserved carbohydrate recognition domains. These characteristic features suggest that ecalectin has unique activities associated with allergic inflammation besides ECA activity. Conversely, ecalectin may mediate ECA activity by binding to a receptor of a known ECA via affinity for the -galactosides present on this receptor. In this study, we have tested whether ecalectin mediates ECA activity by binding to a receptor of a known ECA, and we have assessed its effects on eosinophils. Ecalectin did not mediate ECA activity by binding to the IL-5R or to CCR3. Also, the ECA activity of ecalectin was mainly chemokinetic. In addition, ecalectin induced concentrationdependent eosinophil aggregation, a marker for eosinophil activation. Ecalectin induced concentration-dependent superoxide production from eosinophils but did not induce degranulation; usually these two events are coupled in eosinophil activation. Moreover, ecalectin directly prolonged eosinophil survival in vitro and did not trigger eosinophils to secrete cytokines that prolong eosinophil survival. These results demonstrate that ecalectin has several unique effects on eosinophils. Therefore, we conclude that ecalectin is a novel eosinophil-activating factor. Presumably, these effects allow ecalectin to play a distinctive role in allergic inflammation. The Journal of Immunology, 2002, 168: 1961–1967.
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resently, the prevalence of allergic diseases is increasing rapidly (1). The worldwide economic impact of asthma is estimated to be greater than that of tuberculosis and HIV/ AIDS combined (Bronchial Asthma Fact Sheet No. 206, December, 1998. http://www.who.int/inffs/en/fact206.html). In addition, allergic rhinitis and eosinophil-associated chronic rhinosinusitis affect approximately one-third of the United States population (2). To develop novel treatments and to prevent allergic diseases, further clarification of the complex mechanisms of allergy is necessary. Eosinophils are key effector cells in allergy in addition to mast cells and basophils (3, 4). Eosinophils induce inflammation by releasing cytokines, lipid mediators, toxic oxygen molecules, and cytotoxic granule proteins. Eosinophils, which represent ⬍5% of the total leukocytes in the peripheral blood of healthy people, can accumulate strikingly at allergic inflammation sites to perform effector functions. Therefore, to clarify the complex mechanisms of allergic inflammation, it is necessary to identify and characterize factors that mobilize eosinophils into allergic inflammation sites, namely eosinophil chemoattractants (ECAs)3 (5). Currently recog-
*Departments of Immunology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905; and †Departments of Immunology and Immunopathology, Kagawa Medical School, Kagawa, Japan Received for publication August 14, 2001. Accepted for publication December 10, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institute of Allergy and Infectious Diseases Grants AI 09728, AT 34486, and AI 34577 and by the Mayo Foundation and Yamada Science Foundation (Osaka, Japan). 2 Address correspondence and reprint requests to Dr. Ryoji Matsumoto, Departments of Immunology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905. E-mail address:
[email protected] 3 Abbreviations used in this paper: ECA, eosinophil chemoattractant; [Ca2⫹]i, intracellular Ca2⫹ concentration; EDN, eosinophil-derived neurotoxin; PAF, platelet-activating factor; PI, propidium iodide.
Copyright © 2002 by The American Association of Immunologists
nized ECAs include chemokines (such as eotaxins), cytokines (such as IL-5), lipid mediators, and products of complement activation. Previously, we examined whether Ag-stimulated T lymphocytes produce an ECA and discovered and cloned a novel eosinophilspecific chemoattractant, designated ecalectin (6 – 8). As expected, purified recombinant ecalectin showed potent eosinophil-specific chemoattractant activity in vitro as well as in vivo. In addition to this characteristic feature, ecalectin differs structurally from known ECAs and belongs to the galectin family. Although we previously described ecalectin as a variant of human galectin-9 due to differences in sequence of both amino acids and nucleotides between ecalectin and human galectin-9 (8, 9), extensive analyses recently revealed that these differences resulted from sequence errors. Thus, ecalectin is equivalent to human galectin-9 (10). Galectins are Ca-independent lectins and are defined by their affinity for -galactosides and by their homologous carbohydrate recognition domains (11). Up to now, 12 members of the galectin family have been discovered in mammals (12–14). Although the functions of galectins, except for -galactoside-binding activity, remained obscure for many years, several important functions have been recently reported. For example, galectin-1 induces apoptosis of thymocytes and activated T lymphocytes in the thymus (14) and in the peripheral lymphatic system (15), respectively. In contrast, galectin-3, formerly called Mac-2 or IgE-binding factor, prevents apoptosis of T lymphocytes (16). These findings suggest that galectins regulate immune responses by controlling apoptosis of T lymphocytes, a major immune regulator cell (17). Also, galectin-3 activates mast cells and basophils by inducing degranulation and modulates neutrophil functions (18). Charcot-Leyden crystal protein, stored in eosinophils and basophils, and found abundantly at allergic inflammation sites, is called galectin-10 due to its -galactoside-binding activity and relative homology to other galectins; its function is still unknown (19). Galectins might be involved in a variety of allergic or immune responses (20). 0022-1767/02/$02.00
1962 Taken together, these considerations suggest that ecalectin exerts unique functions in allergy through its own pathways. Thus, a better understanding of the functions of ecalectin may lead to clarification of the complex mechanism of allergy. Conversely, these aspects also suggest that ecalectin mediates ECA activity by binding to a receptor of a known ECA via affinity for -galactosides that are present on this receptor. For example, if the IL-5R has -galactoside residues, ecalectin may bind to and stimulate it. In this example, ecalectin would induce the same eosinophil responses as IL-5 by binding to and stimulating the IL-5R. If so, although ecalectin is structurally different from IL-5, it would be functionally equivalent to IL-5. Therefore, we first performed an Ab-blocking study to determine whether ecalectin mediates ECA activity by binding to a receptor of a known ECA. Then, we examined whether ecalectin has various unique effects on eosinophils. If ecalectin mediates ECA activity through its own pathways and if ecalectin has various unique effects on eosinophils, ecalectin may play a distinctive role in allergic inflammation.
Materials and Methods Reagents Recombinant human eotaxin, IL-3, GM-CSF, and anti-human CCR3 mAb (clone 61828.111) were purchased from R&D Systems (Minneapolis, MN). Anti-human CD18 mAb (clone L130) was bought from BD PharMingen (San Diego, CA). Recombinant human IL-5 and anti-IL-5R ␣-chain mAb (clone KM8400) were generously provided by ScheringPlough (Kenilworth, NJ) and Kyowa Hakko Kogyo (Tokyo, Japan), respectively. The mAbs to IL-5 (clone JS1-5A10), IL-3 (clone BVD8-3G11), and GM-CSF (clone BVD2-23B6) were produced in our laboratory from hybridoma cell lines donated by J. S. Abrams (DNAX, Palo Alto, CA), as described previously (20). Lactose, lactose agarose, Triton X-100, 2-ME, PIPES, Percoll, HEPES, cytochrome c, EDTA, PMSF, pepstatin A, and leupeptin were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640 media and phenol-free RPMI 1640 media were purchased from Celox (Minneapolis, MN).
Production and purification of recombinant ecalectin Recombinant ecalectin was expressed in Spodoptera frugiperda 9 insect cells using baculovirus and was purified, as described previously (8), with slight modifications. In brief, after 4 days of culture, 200 ml of infected S. frugiperda 9 cells were collected by centrifugation and suspended in 40 ml of ice-cold PBS containing 1% Triton X-100, protease inhibitors (100 g/ml PMSF, 1 g/ml pepstatin A, 2 g/ml leupeptin, 5 mM EDTA), and 2-ME (14 mM). The cell suspension was sonicated on ice and was centrifuged to remove debris; the resulting supernatant was applied to a 1-ml lactose agarose column. After washing, ecalectin was eluted with PBS containing 40 mM lactose and 1 mM 2-ME.
Cell preparation Eosinophils were purified from peripheral blood obtained from normal volunteers, as described by Hansel et al. (21), with minor modifications. In brief, heparinized venous blood was layered onto 1.085 g/ml Percoll made in PIPES buffer (25 mM PIPES, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose, pH 7.4) and centrifuged at 1000 ⫻ g in Beckman CS-6KR (Beckman Coulter, Fullerton, CA) for 30 min. After plasma, mononuclear cells, and Percoll layers were removed, erythrocytes were lysed by osmotic shock. The remaining eosinophil/neutrophil pellet was mixed with antiCD16-bound immunomagnetic beads (Miltenyi Biotec, Auburn, CA) and incubated for 1 h. The cells were then separated using a magnetic cell separation system (MACS; Miltenyi Biotec). After the eluate was collected, cell number and eosinophil purity were determined. The purity of eosinophils counted by Randolph’s staining was ⬎97% and the viability was ⬎98%. All the isolation procedures were performed at 4°C or on ice. Neutrophils and PBMCs obtained during this eosinophil purification were used in some experiments.
Analysis of chemotactic and chemokinetic activities Eosinophil migration through a membrane was examined using a 24-well Transwell insert system (Costar, Cambridge, MA) (22). These inserts with porous bottoms (pore, 3 m) serve as the upper chambers, and ordinary tissue culture plate wells serve as the lower chambers. One hundred microliters of the eosinophil suspension (1 ⫻ 106/ml) was added to the upper
ECALECTIN IS AN EOSINOPHIL-ACTIVATING FACTOR chambers, and 600 l of stimuli was added to the lower and upper chambers in a checkerboard titration. The cells and stimuli were suspended in RPMI 1640 medium supplemented with 10% FCS. After a 2-h incubation at 37°C and 5% CO2, the cells that migrated to the lower chamber were collected and counted with light microscopy. In some experiments, eosinophils were preincubated with anti-IL-5R ␣-chain mAb (10 g/ml) or antiCCR3 mAb (10 g/ml) for 30 min at 37°C or 4°C. To examine whether the effects of ecalectin are mediated through binding between galectin and -galactosides (10, 11), 30 mM lactose, a -galactoside, was added to both the lower and upper chambers. For the same reason, lactose was used in the other experiments described below.
Eosinophil aggregation One hundred microliters of freshly isolated eosinophils (1 ⫻ 106/ml) were cultured in a 96-well flat-bottom tissue culture plate (BD Biosciences, Lincoln Park, NJ) with 100 l of serially diluted ecalectin in RPMI 1640 medium supplemented with 10% FCS at 37°C and 5% CO2. After 1, 3, 6, 24, and 48 h of culture, the extent of eosinophil aggregation was observed with an inverted microscope. To determine the effect of lactose, we added lactose (30 mM) to the eosinophil cultures. Similarly, to examine the involvement of divalent cations and Mac-1 (CD11b/CD18) in ecalectin-induced aggregation, we added 5 mM EDTA and anti-CD18 mAb (10 g/ml) to the eosinophil cultures, respectively. Also, neutrophils and PBMCs were cultured with ecalectin and examined.
Measurement of cytosolic-free Ca2⫹ Real-time changes in cytosolic-free Ca2⫹ (intracellular Ca2⫹ concentration ([Ca2⫹]i)) were measured in a flow cytometer using the fluorescent calcium indicator indo-1 (23). This indicator was loaded by incubating 1 ml of eosinophils at 5–10 ⫻ 106/ml with 3 M indo-1/AM (Molecular Probes, Eugene, OR) in phenol red-free RPMI 1640 medium supplemented with 0.1% HSA and 10 mM HEPES for 30 min at 37°C. After washing twice, cells were suspended in the same medium at 5 ⫻ 105 cells/ml. To measure [Ca2⫹]i, cells were stimulated with ecalectin (1 M and 100, 10, and 1 nM) or eotaxin (10 nM), and fluorescence was analyzed by a FACS analyzer equipped with an ion-argon laser (BD Biosciences). [Ca2⫹]i was monitored on the basis of the ratio of the fluorescence of the calcium-bound indo-1 emission (404 nm) and the free indo-1 emission (485 nm).
Superoxide production Eosinophil superoxide production was measured by superoxide dismutaseinhibitable reduction of cytochrome c, as previously reported (24). Freshly isolated eosinophils were suspended in HBSS with 10 mM HEPES, 0.1% gelatin, and 100 M cytochrome c at 5 ⫻ 105 cells/ml. Cell suspension (100 l) was added to the wells of flat-bottom 96-well tissue culture plates. Immediately after addition of stimuli, the absorbance at 550 nm was measured in a microtiter plate autoreader (Thermomax; Molecular Devices, Menlo Park, CA), followed by repeated readings at 37°C. The superoxide anion extinction coefficient of 21.1 ⫻ 103 cm⫺1M⫺1 for reduced cytochrome c at 550 nm was used to express the response as nanomoles of superoxide produced per 105 cells. To examine the effect of lactose on superoxide production by ecalectin, 30 mM lactose was added to the eosinophil cultures. Similarly, we examined whether ecalectin stimulates superoxide production from neutrophils and PBMCs.
Eosinophil degranulation Freshly isolated eosinophils were incubated for 180 min at 37°C and 5% CO2 with stimuli. After incubation, supernatants were collected and stored at ⫺20°C until assayed. To quantitate eosinophil degranulation, the concentration of eosinophil-derived neurotoxin (EDN) in the sample supernatants was measured by RIA. The RIA is a double-Ab competition assay in which radioiodinated EDN, rabbit anti-EDN, and burro anti-rabbit IgG were used, as reported elsewhere (24).
Eosinophil survival assay Freshly isolated eosinophils in suspensions (100 l of 1 ⫻ 106 cells/ml in RPMI 1640 medium supplemented with 10% FCS) were cultured in 96well flat-bottom tissue culture plates for 3 or 4 days at 37°C and 5% CO2 with 100 l of serially diluted ecalectin. As a positive control, 5 pM IL-5 was used. Eosinophil viability was measured as follows. Cultured cells were removed from each well by gentle pipetting and were transferred to 12 ⫻ 75-mm polystyrene round-bottom tubes. An equal volume (200 l) of propidium iodide (PI) solution was added to the cell suspensions to provide a final concentration of 0.50 g/ml PI. At least 10,000 cells from each sample were counted by flow cytometry (FACScan; BD Biosciences), and viable cells were calculated as the percentage of intact cells not stained
The Journal of Immunology with PI divided by the total number of cells (25). To examine cytokines involved in eosinophil survival, mAbs (10 g/ml) to IL-5, IL-3, or GMSCF and 5 pM IL-3 or GM-CSF were used. Also, lactose (30 mM) was added in some experiments.
1963 reported previously, IL-5- or eotaxin-stimulated eosinophils became adherent, resembling fibroblasts (24); ecalectin-stimulated eosinophils did not (data not shown). These findings also suggest that ecalectin acts differently on eosinophils than IL-5 and eotaxin.
Statistical analysis Data are presented as mean ⫾ SEM with the numbers of experiments indicated. Statistical significance was assessed with the paired Student’s t test (InStat Software, San Diego, CA).
Results Ecalectin most likely does not mediate ECA activity by binding to the IL-5R or to the eotaxin receptor, CCR3 Because ecalectin belongs to galectins and thus binds to -galactosides, ecalectin may mediate ECA activity by binding to -galactosides on a known ECA receptor and by stimulating the receptor. We tested this possibility using the IL-5R and the eotaxin receptor, CCR3. As shown in Fig. 1A, the ECA activity of ecalectin was not inhibited by the preincubation of eosinophils with anti-IL-5R ␣-chain mAb or with anti-CCR3 mAb, whereas these Abs did suppress eosinophil migration induced by IL-5 or eotaxin, respectively. In contrast, the ECA activity of ecalectin was inhibited by the addition of lactose, which did not affect eosinophil migration with IL-5 or eotaxin (Fig. 1B). Therefore, ecalectin most likely did not mediate ECA activity through the IL-5R or through CCR3. Furthermore, the binding of ecalectin to -galactosides was important for the interaction between ecalectin and its unidentified receptor. In addition, morphological differences were noted in the eosinophils that migrated to the bottoms of the lower chambers. As
FIGURE 1. Ecalectin does not mediate ECA activity through the IL-5R or CCR3. A, An ECA assay was performed using Transwell plates with a pore size of 3 m. Into the lower chambers and upper chambers, 600 l of factors and 100 l of eosinophil suspension (1 ⫻ 106/ml) were added, respectively. As the ECA factors, ecalectin (1 M, 333 nM, 111 nM, 37 nM), IL-5 (0.5 nM), and eotaxin (10 nM) were used. Eosinophils were preincubated with anti-IL-5R ␣-chain and anti-CCR3 mAb (10 g/ml). After 2 h of incubation, transmigrated cells into the lower chambers were collected and counted under a light microscope. The results depicted are mean ⫾ SD of three experiments performed in duplicate. ⴱ, Statistically significant difference (p ⬍ 0.05) compared with cells with medium alone. †, Statistically significant difference (p ⬍ 0.05) compared with cells stimulated with IL-5. ††, Statistically significant difference (p ⬍ 0.05) compared with cells stimulated with eotaxin. B, Similarly, transmigrated cells into the lower chambers with the stimuli in the presence of lactose (30 mM) were counted. The results depicted are mean ⫾ SD of three experiments performed in duplicate. ⴱⴱ, Statistically significant difference (p ⬍ 0.05) compared with cells stimulated with ecalectin (111 nM).
Effects of ecalectin on eosinophils are mainly chemokinetic We examined whether the ECA activity of ecalectin is chemokinetic like IL-5 or chemotactic like eotaxin. As shown in Table I, eosinophil migration through the membrane’s pores was mainly independent of ecalectin gradients. These results suggest that ECA activity of ecalectin is mainly chemokinetic. Ecalectin induces eosinophil aggregation Fig. 1 and Table I show that high concentrations of ecalectin inhibit eosinophil migration. For example, fewer eosinophils migrated in the presence of 1 M ecalectin than with medium alone. By inverted microscopy, we found that this inhibition was caused by a marked aggregation of eosinophils on the membrane’s pores of the upper chamber (data not shown). To further investigate eosinophil aggregation induced by ecalectin, eosinophils were incubated with ecalectin for 1, 3, 6, 24, and 48 h. As shown in Fig. 2, ecalectin induced eosinophil aggregation concentration dependently, but the addition of 30 mM lactose inhibited this aggregation (data not shown). Next, because eosinophil aggregation in guinea pigs is induced by a Ca2⫹-dependent interaction between Mac-1 (CD11b/CD18) and ICAM-1, which are expressed on eosinophils after stimulation (26, 27), we examined the effects of
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ECALECTIN IS AN EOSINOPHIL-ACTIVATING FACTOR Table I. Checkerboard analysis of eosinophil migration with ecalectina Ecalectin in Lower Chamber Ecalectin in Upper Chamber (nM)
0 50 (nM) 150 (nM) 450 (nM)
0
50 nM
150 nM
450 nM
5.4 ⫾ 1.1 15.8 ⫾ 4.4 19.4 ⫾ 3.9 9.9 ⫾ 2.6
27.5 ⫾ 3.8 34.2 ⫾ 4.1 23.4 ⫾ 2.9 13.1 ⫾ 2.4
45.1 ⫾ 3.9 29.3 ⫾ 2.8 26.1 ⫾ 3.6 6.3 ⫾ 2.9
14.1 ⫾ 2.5 14.4 ⫾ 2.9 6.3 ⫾ 2.4 3.2 ⫾ 1.6
a Various concentrations of ecalectin (0, 50, 150, 450 nM) were added to the lower or upper chambers or both. Numbers of eosinophils (⫻103) migrated through 3-m pores into the lower chambers are shown as mean ⫾ SD from three experiments.
EDTA and anti-CD18 mAb on ecalectin-induced eosinophil aggregation. Ecalectin-induced aggregation was inhibited by EDTA but not by anti-CD18 mAb (data not shown). These results suggest that ecalectin-induced eosinophil aggregation, which needs divalent cations, is not related to interaction between Mac-1 (CD11b/ CD18) and ICAM-1. Similar tests of ecalectin with purified neutrophils and PBMCs showed no aggregation (data not shown). Ecalectin induces superoxide production but not degranulation Next, we examined whether ecalectin activates eosinophils. As shown in Fig. 3, ecalectin induced a marked concentration-dependent superoxide production from eosinophils. This ecalectin-induced superoxide production was also inhibited by lactose. However, as shown in Fig. 4, ecalectin did not induce eosinophil degranulation. This is striking in contrast to the marked degranulation induced by IL-5 and in contrast to previous results showing that superoxide production and degranulation are usually coupled (24). Taken together, these results suggest the existence of separate pathways for superoxide production and degranulation. In addition, although eotaxin (10 nM) induced a marked Ca2⫹ influx in eosinophils, ecalectin (1 M and 100, 10, and 1 nM) did not (data not shown). This is a striking contrast, because seven-transmembrane receptors (for example, receptors of chemokines, lipid mediators, and complement products) always induce a Ca2⫹ influx after ligand binding (28, 29). Consequently, ecalectin evidently activates eosinophils through a novel pathway. Ecalectin prolongs eosinophil survival in vitro Some galectins regulate apoptosis of lymphocytes (14 –17). In addition, cytokines with ECA activity (IL-5, IL-3, and GM-CSF) prevent apoptosis of eosinophils and thus prolong eosinophil survival in vitro (30 –33). Therefore, to examine the effect of ecalectin on apoptosis of eosinophils, we investigated whether ecalectin prolongs eosinophil survival in vitro. As shown in Fig. 5A, ecalectin prolonged eosinophil survival concentration dependently. Next, because fibronectin (32) and other molecules (25) prolong eosin-
ophil survival indirectly by triggering eosinophils to secrete cytokines that prolong eosinophil survival, we examined whether this effect of ecalectin was direct. As shown in Fig. 5B, eosinophil survival prolonged by ecalectin was not inhibited by mAbs to IL-5, IL-3, or GM-CSF, but by lactose (30 mM). When we increased the concentration of ecalectin, the eosinophil survival was not increased (data not shown). These results suggest that ecalectin directly prolongs eosinophil survival in vitro through a pathway different from those of IL-3, IL-5, and GM-CSF, and, thus, probably through a novel mechanism.
Discussion In this study, we demonstrated that ecalectin has unique effects on eosinophils, and, thus, ecalectin is a novel eosinophil-activating factor. We originally speculated that ecalectin mediates ECA activity by binding to -galactosides on a receptor of a known ECA via affinity for -galactosides, and thus stimulates the receptor. Because detailed carbohydrate structures on these receptors are not yet available, it is unclear which ECA receptors have -galactoside residues. However, most extracellular proteins (including the extracellular portions of receptors) are glycosylated and theoretically have -galactoside residues (35, 36). Thus, most ECA receptors may have -galactoside residues. To examine whether ecalectin binds to and stimulates a receptor of a known ECA, we performed an Ab-blocking study in an ECA assay. We selected two key receptors on eosinophils and used two Abs (anti-IL-5R ␣-chain mAb and anti-CCR3 mAb) for blocking. Clearly, numerous receptors could be tested, but we tested these two for the following reasons. First, because the ECA activity of ecalectin is specific for eosinophils, we chose the IL-5/IL-5R and the eotaxin/CCR3 pathways, both of which are relatively specific for human eosinophils (37, 38). In contrast, we also recognize that the IL-5/IL-5R pathway is used by B cells in mice and the eotaxin/CCR3 pathway is used by Th2-type Th lymphocytes and basophils (39). Second, because the increased expression of IL-5 and eotaxin is often observed at sites of eosinophil accumulation, these two factors are
FIGURE 2. Ecalectin induces concentration-dependent eosinophil aggregation. Eosinophils were cultured for 48 h with serially diluted ecalectin (A, 0; B, 1 M; C, 333 nM; D, 111 nM). Concentration-dependent aggregation with ecalectin was observed with an inverted microscope (⫻200). Ecalectininduced aggregation was observed after 1 h of culture, and the extent of aggregation increased with longer times. Because the maximal aggregation was observed after 24 h of culture, we show eosinophil aggregation after 24 h of culture.
The Journal of Immunology
FIGURE 3. Effect of ecalectin on superoxide anion generation. Eosinophils were incubated with various concentrations of ecalectin (200, 150, 100, and 50 nM), ecalectin (200 nM) in the presence of lactose (30 mM) or IL-5 (0.5 nM). Kinetics of superoxide production was measured by reduction of cytochrome c, as described in Materials and Methods. Results were presented as means ⫾ SD from five experiments. ⴱ, Statistically significant difference (p ⬍ 0.05) compared with cells stimulated with ecalectin (200 nM).
regarded as the most important ECAs. Third, if we classify ECAs into two types, the features of these two factors represent two types of ECAs. The cytokine, IL-5, is a chemokinetic ECA and signals through a typical cytokine receptor without a Ca2⫹ influx. In contrast, the chemokine, eotaxin, is a chemotactic ECA and signals through a typical seven-transmembrane receptor with a Ca2⫹ influx (lipid mediators and products of complement activation use seven-transmembrane receptors and, thus, may be included in this eotaxin group). Finally, although we did not test all other ECAs, we proceeded to the functional studies to show indirectly that ecalectin does not signal through the receptors for other ECAs. Ecalectin did not seem to activate eosinophils through the receptors for other ECAs; ecalectin may bind to them but does not stimulate them. In the functional studies, we found other distinctive activities of ecalectin that support the independence of ecalectin from known ECA receptors. That is, if ecalectin did mediate its activities through a known ECA receptor, the activities of ecalectin would be the same as this known ECA. Also, because ecalectin may bind to, but not stimulate, receptors for known ECAs, the functional studies could compensate for limitations in the Ab-blocking study. Indeed, the functional studies revealed that no ECA has the same activities as ecalectin. Thus, ecalectin probably does not use a receptor for other ECAs for signal transduction, but mediates its activities through its own pathway, namely through binding to its own, as yet unidentified, receptor. Because all of ecalectin’s activities tested were inhibited by lactose, interaction with -galactosides is important for the binding of ecalectin to its receptor. As shown by functional and structural analyses, each galectin probably has a fine specificity for sugar moieties to which it binds (40). Thus, ecalectin seemingly binds to its receptor molecule expressed
1965
FIGURE 4. Effect of ecalectin on eosinophil degranulation. Eosinophils were incubated for 3 h with various concentrations of ecalectin (200, 150, 100, and 50 nM) or IL-5 (0.5 nM). After incubation, supernatants were collected and stored at ⫺20°C until assayed. The amounts of EDN released from the supernatants were measured by RIA. Results are represented as means ⫾ SD from four experiments. ⴱ, Statistically significant difference (p ⬍ 0.05) compared with cells with medium alone.
on eosinophils, and this receptor most likely has specific sugar moieties. Because ecalectin does not induce a Ca2⫹ influx into eosinophils, the ecalectin receptor seems to be quite different from the receptors of most ECAs (chemokines, lipid mediators, complement products), which are seven-transmembrane receptors and induce a Ca2⫹ influx after the ligand binding (28, 29). Conversely, the ecalectin receptor may be related to a cytokine receptor that does not induce a Ca2⫹ influx. Galectin-binding molecules have been studied in other galectins (41– 43). In these studies, galectins showed considerable specificity, even though they had been expected to bind to many glycoproteins by affinity for -galactosides; most glycoproteins theoretically contain -galactosides, as described above. For example, galectin-1 binds to a few kinds of glycoproteins on T cells, but this binding seems to stimulate only one kind of molecule, namely CD45 (41, 42). Although it is unclear that CD45 is the proper receptor for galectin-1, we suspect that ecalectin also binds to and stimulates one type of molecule expressed on eosinophils. Considering the effects of ecalectin on eosinophils, this ecalectin receptor molecule is probably expressed only on eosinophils and not on other cells. Alternatively, although the ecalectin receptor may be expressed on other cells, the glycosylation state of this molecule may be different in eosinophils. This hypothesis may be reasonable, because we did notice variability in the in vitro bioassays of ecalectin among the different eosinophil donors tested (data not shown). Also, others have reported that the interaction between galectin-1 and CD45 may be modulated by the glycosylation state of CD45 (42). However, we cannot exclude the following two possibilities. Ecalectin may bind to a receptor for another ECA at the site to which the original ligand does not bind and could stimulate the receptor. In this case, the receptor could signal via other pathways to perform different effector functions. Alternatively, ecalectin may bind to several different receptors for other ECAs. Even if these ecalectin binding sites are not the original ligand binding sites, ecalectin could cross-link these
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ECALECTIN IS AN EOSINOPHIL-ACTIVATING FACTOR
FIGURE 5. Ecalectin prolongs eosinophil survival in vitro. A, Eosinophils were cultured with ecalectin (10, 3.3, and 1 nM) or IL-5 (5 pM). After 1, 2, and 3 days of culture, the ratio of viable eosinophils was counted by FACS analysis. The cells incorporating PI were regarded as dying or dead cells. At least 10,000 cells were analyzed from each sample. Results are presented as mean percentage of viable cells ⫾ SD from four separate experiments. B, From the FACS analysis used in A, we examined whether eosinophil survival is prolonged by IL-5, IL-3, or GM-CSF secreted from eosinophils after stimulation with ecalectin. Also, we examined the effect of lactose (30 mM) on eosinophil survival prolonged by IL-5 and ecalectin. The assay was performed on day 4. The concentrations of the cytokines, Abs, and ecalectin were 5 pM, 10 g/ml, and 10 nM, respectively. Results are presented as mean percentage of viable cells ⫾ SD from four separate experiments. ⴱ, Statistically significant difference (p ⬍ 0.05) compared with cells with medium alone. ⴱⴱ, Statistically significant difference (p ⬍ 0.05) compared with cells stimulated with ecalectin. †, Statistically significant difference (p ⬍ 0.05) compared with cells stimulated with IL-5. ††, Statistically significant difference (p ⬍ 0.05) compared with cells stimulated with IL-3. †††, Statistically significant difference (p ⬍ 0.05) compared with cells stimulated with GM-CSF.
several different receptors, and this cross-linking of different receptors might result in distinctive signaling. From the chemokinetic and aggregation activities of ecalectin, we suspected that ecalectin would activate eosinophils. Other chemokinetic ECAs, e.g., IL-5, activate eosinophils and induce superoxide production and degranulation (44). Also, eosinophil aggregation after stimulation with platelet-activating factor (PAF) is another marker of eosinophil activation in guinea pigs (26). In humans, only PMA induces direct aggregation of eosinophils, but both aggregated and degranulated eosinophils are observed at allergic inflammation sites, indicating eosinophil activation (27). Indeed, ecalectin did activate eosinophils, as shown by concentration-dependent superoxide production. Surprisingly, however, ecalectin induced very limited eosinophil degranulation. Because superoxide production and degranulation are usually coupled in eosinophil activation (24), these unusual effects of ecalectin suggest the existence of separate pathways for superoxide production and degranulation. These effects may be related to morphological changes of eosinophils; for degranulation, eosinophils need to be adherent, whereas ecalectin-stimulated eosinophils are not. In regard to ecalectin-induced eosinophil aggregation, we suggest three possible mechanisms. The first mechanism is through an interaction between Mac-1 (CD11b/CD18) and ICAM-1. For example, because ICAM-1 is newly expressed on the eosinophil’s surface after PAF stimulation, guinea pig eosinophil aggregation after stimulation with PAF is induced by a Ca2⫹-dependent interaction between Mac-1 (CD11b/CD18) and ICAM-1 (26, 27). Inhibition of aggregation by EDTA is consistent with this first mechanism, but the lack of inhibition by anti-CD18 mAb that prevents Mac-1 (CD11b/CD18) and ICAM-1 interaction is not. The second mechanism is related to hemagglutination induced by galectins (10, 45). Hemagglutination in rabbit erythrocytes is due to interaction between galectins and -galactoside residues on these erythrocytes via the -galactoside-binding activity of galectins.
Accordingly, this hemagglutination is Ca2⫹ independent and thus is not inhibited by EDTA. Although divalent cations may be required for dimerization or polymerization of prototype galectins (46), which have one carbohydrate binding site, to induce hemagglutination, ecalectin is a tandem-type galectin and (without dimerization or polymerization) has two carbohydrate binding sites for -galactosides per molecule (47). Thus, divalent cations would not be required for ecalectin-induced hemagglutination. As a third mechanism, we hypothesize that, after binding to its unidentified receptor on eosinophils, ecalectin induces expression of a certain surface molecule. This newly expressed molecule does need a divalent cation to bind to its counterligand or receptor on eosinophils similar to the interaction between Mac-1 (CD11b/CD18) and ICAM-1. Although its precise mechanism remains to be elucidated, this aggregation activity also emphasizes the uniqueness of the activity of ecalectin. Ecalectin prolonged eosinophil survival in vitro by preventing apoptosis of eosinophils. We anticipated this result based on two previous findings on apoptosis. First, certain galectins induce or prevent apoptosis of lymphocytes (14 –17). Second, three ECAs (IL-5, IL-3, and GM-CSF) prolong eosinophil survival in vitro by preventing apoptosis of eosinophils (30 –33). In addition, fibronectin and other molecules prevent apoptosis by triggering eosinophils to secrete these cytokines in an autocrine mechanism (25, 34). Accordingly, because the receptors for IL-5, IL-3, and GM-CSF, with their ligand-specific ␣-chain and common -chain, transduce their signals through the common -chain (48), prolonged eosinophil survival is mediated through the common -chain pathway. However, in contrast to fibronectin and other molecules, the activity of ecalectin is not mediated through this common -chain pathway, triggering autocrine secretion of IL-5, IL-3, or GM-CSF, but directly through its own pathway and most likely through a novel mechanism. Because eosinophils are end-stage cells, they do
The Journal of Immunology not proliferate and are destined for apoptosis. Thus, prolonged eosinophil survival allows eosinophils to perform their effector functions in tissues for extended times. Further investigation of the novel mechanism of ecalectin is important and may help to elucidate the mechanisms of galectin-regulated apoptosis. In conclusion, this study shows that ecalectin is a novel eosinophil-activating factor. Ecalectin has chemokinetic ECA activity and induces concentration-dependent eosinophil aggregation. Ecalectin activates eosinophils, as shown by superoxide production, but ecalectin does not induce degranulation, and ecalectin prolongs eosinophil survival directly. Therefore, although the receptor for ecalectin is still unknown, these effects allow ecalectin to play a distinctive role in allergic inflammation.
Acknowledgments We thank Cheryl Adolphson for her editorial assistance and Linda H. Arneson for her excellent secretarial assistance. We also are grateful to James E. Tarara and Kathleen R. Bartemes for their technical assistance in flow cytometry and the EDN assay, respectively.
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