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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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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
concentration-dependent 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 recognized 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 eosinophil-specific chemoattractant, designated ecalectin (6, 7, 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, 13, 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).
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 Schering-Plough
(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 µg/ml PMSF, 1 µg/ml pepstatin A, 2 µg/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 x 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 anti-CD16-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 µm)
serve as the upper chambers, and ordinary tissue culture plate wells
serve as the lower chambers. One hundred microliters of the eosinophil
suspension (1 x 106/ml) was added to the
upper chambers, and 600 µl 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 µg/ml) or anti-CCR3 mAb (10
µg/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 x 106/ml) were cultured in a 96-well flat-bottom tissue culture plate (BD Biosciences, Lincoln Park, NJ) with 100 µl 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 µg/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 x 106/ml with 3 µM 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 x 105 cells/ml. To measure [Ca2+]i, cells were stimulated with ecalectin (1 µM 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 dismutase-inhibitable reduction of cytochrome c, as previously reported (24). Freshly isolated eosinophils were suspended in HBSS with 10 mM HEPES, 0.1% gelatin, and 100 µM cytochrome c at 5 x 105 cells/ml. Cell suspension (100 µl) 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 x 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 µl of 1 x 106 cells/ml in RPMI 1640 medium supplemented with 10% FCS) were cultured in 96-well flat-bottom tissue culture plates for 3 or 4 days at 37°C and 5% CO2 with 100 µl 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 x 75-mm polystyrene round-bottom tubes. An equal volume (200 µl) of propidium iodide (PI) solution was added to the cell suspensions to provide a final concentration of 0.50 µg/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 with PI divided by the total number of cells (25). To examine cytokines involved in eosinophil survival, mAbs (10 µg/ml) to IL-5, IL-3, or GM-SCF and 5 pM IL-3 or GM-CSF were used. Also, lactose (30 mM) was added in some experiments.
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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
A, 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 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.
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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.
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Fig. 1
and Table I show that high concentrations of ecalectin
inhibit eosinophil migration. For example, fewer eosinophils migrated
in the presence of 1 µM 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 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).
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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
µM 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.
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Some galectins regulate apoptosis of lymphocytes
(14, 15, 16, 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, 31, 32, 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 eosinophil 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-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 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 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, 42, 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 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, 15, 16, 17). Second, three
ECAs (IL-5, IL-3, and GM-CSF) prolong eosinophil survival in vitro by
preventing apoptosis of eosinophils (30, 31, 32, 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 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.
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Acknowledgments |
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Footnotes |
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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: matsumoto.ryoji{at}mayo.edu
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.
Received for publication August 14, 2001. Accepted for publication December 10, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactoside binding lectin. J. Clin. Invest. 99:2452.[Medline]
RII (CD32) pivotally regulates survival of human eosinophils. J. Immunol. 162:4253.This article has been cited by other articles:
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 L. V Norling, M. Perretti, and D. Cooper Endogenous galectins and the control of the host inflammatory response J. Endocrinol., May 1, 2009; 201(2): 169 - 184. [Abstract] [Full Text] [PDF]
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 A. Matsuura, J. Tsukada, T. Mizobe, T. Higashi, F. Mouri, R. Tanikawa, A. Yamauchi, M. Hirashima, and Y. Tanaka Intracellular galectin-9 activates inflammatory cytokines in monocytes. Genes Cells, April 1, 2009; 14(4): 511 - 521. [Abstract] [Full Text] [PDF]
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 K. Nagahara, T. Arikawa, S. Oomizu, K. Kontani, A. Nobumoto, H. Tateno, K. Watanabe, T. Niki, S. Katoh, M. Miyake, et al. Galectin-9 Increases Tim-3+ Dendritic Cells and CD8+ T Cells and Enhances Antitumor Immunity via Galectin-9-Tim-3 Interactions J. Immunol., December 1, 2008; 181(11): 7660 - 7669. [Abstract] [Full Text] [PDF]
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N. Farahi, A. S. Cowburn, P. D. Upton, J. Deighton, A. Sobolewski, E. Gherardi, N. W. Morrell, and E. R. Chilvers Eotaxin-1/CC Chemokine Ligand 11: A Novel Eosinophil Survival Factor Secreted by Human Pulmonary Artery Endothelial Cells J. Immunol., July 15, 2007; 179(2): 1264 - 1273. [Abstract] [Full Text] [PDF]
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 S. Katoh, N. Ishii, A. Nobumoto, K. Takeshita, S.-Y. Dai, R. Shinonaga, T. Niki, N. Nishi, A. Tominaga, A. Yamauchi, et al. Galectin-9 Inhibits CD44-Hyaluronan Interaction and Suppresses a Murine Model of Allergic Asthma Am. J. Respir. Crit. Care Med., July 1, 2007; 176(1): 27 - 35. [Abstract] [Full Text] [PDF]
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 J M Ilarregui, G A Bianco, M A Toscano, and G A Rabinovich The coming of age of galectins as immunomodulatory agents: impact of these carbohydrate binding proteins in T cell physiology and chronic inflammatory disorders Ann Rheum Dis, November 1, 2005; 64(suppl_4): iv96 - iv103. [Abstract] [Full Text] [PDF]
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S.-Y. Dai, R. Nakagawa, A. Itoh, H. Murakami, Y. Kashio, H. Abe, S. Katoh, K. Kontani, M. Kihara, S.-L. Zhang, et al. Galectin-9 Induces Maturation of Human Monocyte-Derived Dendritic Cells J. Immunol., September 1, 2005; 175(5): 2974 - 2981. [Abstract] [Full Text] [PDF]
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 W.-C. Chou, H.-Y. Chen, S.-L. Yu, L. Cheng, P.-C. Yang, and C. V. Dang Arsenic suppresses gene expression in promyelocytic leukemia cells partly through Sp1 oxidation Blood, July 1, 2005; 106(1): 304 - 310. [Abstract] [Full Text] [PDF]
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H. Asakura, Y. Kashio, K. Nakamura, M. Seki, S. Dai, Y. Shirato, M. J. Abedin, N. Yoshida, N. Nishi, T. Imaizumi, et al. Selective Eosinophil Adhesion to Fibroblast Via IFN-{gamma}-Induced Galectin-9 J. Immunol., November 15, 2002; 169(10): 5912 - 5918. [Abstract] [Full Text] [PDF]
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J. D. Hernandez and L. G. Baum Ah, sweet mystery of death! Galectins and control of cell fate Glycobiology, October 1, 2002; 12(10): 127R - 136R. [Abstract] [Full Text] [PDF]
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