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IMMUNOBIOLOGY
From the Pulmonary Research Group, Department of
Medicine, University of Alberta, Edmonton, Canada; and Division of
Respirology, University of Toronto, ON, Canada.
The tetraspanin CD63 (also known as LAMP-3) has been
implicated in phagocytic and intracellular lysosome-phagosome
fusion events. It is also present in eosinophils, with
predominant expression on crystalloid granule membrane. However, its
role in eosinophil function is obscure. We hypothesized that CD63
is associated with intracellular events involved in eosinophil
activation and mediator release. We used a combination of confocal
immunofluorescence microscopy, flow cytometry, and secretion assays,
including Eosinophils are major effector cells in allergic
inflammation and asthma.1-3 They synthesize, store, and
release a wide range of proinflammatory mediators, including at least 4 cationic proteins1,2 and up to 23 cytokines and growth
factors.4,5 Eosinophils contain different populations of
mediator-storage organelles, including small secretory vesicles as well
as crystalloid granules. The latter secretory granules are the site of
storage of cytotoxic cationic proteins as well as a number of
cytokines, chemokines, and growth factors.6,7 The
membrane-bound crystalloid granule comprises 2 compartments: an
electron-dense crystalline core (internum) where major basic protein
(MBP)8,9 is stored and an electron-lucent matrix6 where 3 cationic proteins In eosinophils, 3 mediator release mechanisms have been described:
cytolysis or necrotic release, compound exocytosis, and piecemeal
degranulation (PMD).13 In cytolysis, the cell membrane loses its integrity and crystalloid granules are released to
extracellular space. In compound exocytosis, a number of granules fuse
intracellularly to form a large degranulation chamber or cavity, which
in turn fuses with the cell membrane before discharging its contents to extracellular space. In physiologic conditions, a more commonly seen
mode of exocytotic mediator release in eosinophils is PMD, whereby
stored mediators are selectively released from an intragranular pool,
leaving portions or all of the granules empty in the intact cell.13,14 Various stimuli, including cross-linking of
different subclasses of immunoglobulin receptors, are known to induce
selective mediator release from eosinophils.15,16 Our own
previous studies have demonstrated that interferon- CD63, also known as lysosome-associated membrane
protein-3,17,18 is a member of the transmembrane-4
superfamily whose membership has grown to 20 proteins since its first
discovery in 1990. Tetraspanins are membrane-associated molecules that
span the membrane 4 times (TM1-TM4) with 2 extracellular domains
(EC1-EC2). As cell surface proteins, tetraspanins appear to act as
"molecular facilitators" by increasing the formation and stability
of functional signaling complexes.19 The tetraspanin CD63
is proposed to be involved in a number of cellular functions such as
cell activation20 and mediator release.21
CD63 is a well-established component of the late endosomal and
lysosomal membranes.22 CD63 is also present in
"secretory lysosomes,"23 the secretory granules of cells derived from the hemopoietic lineage that are related to lysosomes. These organelles include azurophilic granules of
neutrophils24 and Present only in myeloperoxidase-containing granules of neutrophils,
CD63 has been described as a marker for azurophilic granule fusion,
with the plasma membrane suggesting a potential role for this molecule
in membrane fusion events.24,26 In the rat basophilic leukemia cell line, an antibody against CD63 (AD1) inhibited
immunoglobulin E (IgE)-mediated histamine release, suggesting a role
for CD63 in events associated with mediator release.27
A number of tetraspanins, including CD9, CD37, CD53, and CD63, are
expressed in peripheral blood eosinophils.28,29
Cross-linking of some surface transmembrane-4 superfamily molecules
induces significant eosinophil homotypic aggregation, up-regulation of CD11b expression, or CD62L shedding, consistent with cellular activation.29 Yet, no function has been attributed to CD63
in human eosinophils, and very little is known about its possible role.
We hypothesized that CD63 is associated with intracellular events
involved in eosinophil activation and mediator release. We have used a
combination of double immunofluorescent staining and confocal laser
scanning microscopy, flow cytometry, Preparation of eosinophils
The study was approved by the Health Research Ethics Board of the
Faculty of Medicine and Dentistry and University of Alberta Hospital.
Informed consent was provided according to the Declaration of Helsinki.
Granule purification
Flow cytometry Eosinophils (5 × 105 cells per test) incubated in the presence or absence of IFN- (20 ng/mL; R&D Systems, Minneapolis,
MN); a combination of C5a (800 nM) and cytochalasin B (CB) (10 ng/mL; Sigma); and a cocktail of IL-3 (10 ng/mL), IL-5 (5 ng/mL), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/mL)
(Genzyme, Cambridge, MA) (10 minutes) were fixed in 5% formalin (10 minutes at 4°C) and blocked in 5% milk in flow buffer (phosphate-buffered saline + 1% bovine serum albumin + 0.1%
sodium azide) overnight at 4°C. Following blocking, cells were washed (× 3) in flow buffer and incubated with 5 µg/mL of one of the following antibodies: mouse monoclonal anti-CD63 (IgG1;
Pharmingen, San Diego, CA) or mouse IgG1 isotype control
(R&D Systems) for 60 minutes on ice. After 3 washing steps, cells were
incubated with goat F(ab')2 antimouse IgG conjugated to
phycoerythrin (5 µg/mL) (Cedarlane Laboratories, Hornby, ON, Canada)
for 30 minutes at 4°C. The cells were subsequently washed 3 times and
resuspended in flow buffer to a final density of
1 × 106/mL and analyzed on a FACScan instrument using
CellQuest software (Becton Dickinson, San Jose, CA). Eosinophil
crystalloid granules obtained from purified eosinophils were subjected
to the same procedure described above and further examined using FACS
analysis. To examine the intracellular or intragranular pool of CD63,
eosinophils or their purified granules were permeabilized by adding
0.1% saponin to the blocking reagent.
Immunofluorescent labeling Cytospins of resting as well as IFN- (20 ng/mL), a
combination of C5a (800 nM) and CB (10 ng/mL), and IL-3 (10 ng/mL)/IL-5 (5 ng/mL)/GM-CSF (10 ng/mL)-stimulated (10 minutes) eosinophils were
prepared by sedimenting 3 × 104 cells (suspended in 100 µL 20% fetal calf serum in RPMI 1640) in a Cytospin 2 Centrifuge
(Shandon, Runcorn, United Kingdom) at 800 rpm for 2 minutes. To
prepare cytospins of crystalloid granules, the same procedure was
carried out on highly purified crystalloid granules. Slides were then
foil-wrapped and stored at  20°C until used. Slides of resting and
agonist-stimulated eosinophils were fixed for 8 minutes in 2%
paraformaldehyde in phosphate-buffered saline (room temperature) and
washed (× 5) in Tris-buffered saline (TBS, pH 7.4). Following
fixation, cytospins were blocked using 3% fetal calf serum in a
humidified container (30 minutes). After a second washing step, slides
were incubated overnight with TBS containing 1% mouse monoclonal
antihuman CD63 (5 µg/mL) at 4°C. BODIPY-FL-conjugated goat
antimouse secondary antibody (20 µg/mL) (Molecular Probes, Eugene,
OR) was used to detect immunoreactivity of CD63 (2 hours, room
temperature). Following another washing step, slides were blocked again
for 2 hours using goat antimouse IgG Fab fragment (Jackson
ImmunoResearch Laboratories, West Grove, PA) (50 µg/mL) and
double-labeled with either mouse monoclonal antihuman MBP (1%)
(BMK-13, generated in-house31) or mouse monoclonal
antihuman RANTES (5 µg/mL) (R&D Systems) at 4°C. Immunoreactivity
against MBP and RANTES was detected by incubating slides with 15 µg/mL rhodamine (TRITC)-labeled goat antimouse secondary antibody
(Jackson ImmunoResearch Laboratories) for 2 hours. Mouse IgG1 (R&D
Systems) at equivalent concentrations was used as the isotype control.
After a final washing step, 10 µL of the antibleaching agent, 0.4%
n-propyl gallate (Sigma) in 3:1 glycerol 10 × TBS, was applied to the
slides before coverslip attachment.
Confocal laser scanning microscopy Immunofluorescent stainings of resting and stimulated eosinophils as well as purified crystalloid granules were examined using a Zeiss laser scanning confocal microscope (LSM 510) mounted on a Zeiss Axiovert M100 inverted microscope with a × 63 plan-apochromatic lens (Zeiss, Toronto, Canada). The 488-nm laser line (generated from a 25-mW argon laser) and 543-nm laser line (generated from 1-mW HeNe laser) were used to image BODIPY-FL (green) and TRITC (red), used in the experiments. A bandpass filter (505-550 nm) was used to collect emission from BODIPY-FL, and a longpass filter (560 nm) was used to collect signals from TRITC. To avoid spillover of the fluorochromes, sequential scanning mode of the machine was used to collect images from double-stained samples. Image acquisition was optimized using the required pinhole setting, photomultiplier gain, and offset. Higher spatial resolution was achieved by using the appropriate zoom on the computer. Images were further analyzed and developed using LSMIB 4.0 software.Measurement of  -hex, freshly purified eosinophils
(2 × 105 cells) were stimulated using IFN-, C5a/CB,
or IL-3/IL-5/GM-CSF for 10 minutes. Cell-free supernatants were
collected, and pellets were lysed using 0.5% Triton X-100 (Sigma) in
color-free RPMI. Samples of cell-free supernatants (50 µL) and lysed
pellets (50 µL) were mixed with 50 µL substrate solution (1 mM
4-methylumbelliferyl N-acetyl--D-glucosaminide in 0.2 M
citrate buffer, pH 4.5, and 0.1% Triton X-100) in a 96-well microplate
and incubated for 60 minutes at 37°C. The reaction was terminated by
the addition of 150 µL ice-cold 0.2 M Tris, and the fluorescence
(excitation 360 nm, emission 460 nm) was measured in a Millipore
CytoFluor 2350 plate reader (Millipore, Nepean, ON, Canada) as
described previously.10,11 For EPO measurement, freshly
purified eosinophils (2 × 105 cells) were stimulated
using IFN- (20 ng/mL). EPO activity in the samples of cell-free
supernatants as well as the cell pellets was assayed using TMB (3,3',
5,5'-tetramethylbenzidine liquid substrate system) solution by
combining 50 µL samples with 150 µL substrate solutions in a
96-well microplate and incubating at room temperature for 30 minutes.
The reaction was terminated by adding 50 µL of 1 M sulfuric acid, and
absorbance was read at 450 nm in a spectrophotometric microplate reader
(Vmax Kinetic Microplate Reader, Molecular Devices, Sunnyvale, CA).
RANTES immunoreactivity in supernatant of IFN--stimulated cells (10 minutes) was measured using a Quantikine enzyme-linked immunosorbent
assay kit (R&D Systems) with a detection sensitivity of 31.2 pg/mL
according to manufacturer's instructions.
Statistical analysis Experiments were done in triplicates. Values were averaged, and their SEM was calculated. Results were analyzed for significance using a Student t test. A P value of less than .01 was considered significant.
CD63 protein expression and localization in human peripheral blood eosinophils The subcellular distribution of CD63 was examined using a combination of immunofluorescent staining and confocal laser scanning microscopy on cytospins of purified peripheral blood eosinophils. Cytospins of freshly purified eosinophils were prepared and immunostained using a monoclonal anti-CD63 antibody. CD63 was expressed in resting peripheral blood eosinophils and appeared predominantly localized to the membrane of all crystalloid granules (Figure 1A). The association of CD63 immunoreactivity with crystalloid granule membrane was further confirmed by immunofluorescent staining of a dispersed population of eosinophil crystalloid granules (Figure 1B).
To determine the intracellular localization site of CD63 relative to MBP (marker for the eosinophil granule crystalline core) and RANTES (marker for the crystalloid granule matrix region), we carried out a double immunofluorescent staining procedure on a population of isolated granules as well as freshly purified eosinophils. While immunoreactivity against MBP predominantly localized to the core region (Figure 1C-D) and did not colocalize with CD63, the immunoreactivity against RANTES and CD63 showed relative colocalization to the peripheral compartment of the crystalloid granules highlighted by the yellow color in the merged image (Figure 1E). Mobilization of CD63 in stimulated eosinophils To examine the intracellular localization and kinetics of CD63 mobilization relative to MBP in agonist-stimulated cells, freshly isolated eosinophils were stimulated (10 minutes) with either of IFN- (20 ng/mL), C5a (800 nM)/CB (10 ng/mL), or a cocktail of IL-3
(10 ng/mL)/IL-5 (5 ng/mL) and GM-CSF (10 ng/mL). Cytospins of
stimulated eosinophils were prepared, double-immunostained for CD63 and
MBP, and examined using confocal fluorescent microscopy. As early as 10 minutes after IFN- (Figure 1F-H) or C5a/CB stimulation (Figure
1I-K), CD63 immunostaining confined to the regions adjacent to the cell
membrane, while MBP immunoreactivity except for some intensification at
the cell periphery remained relatively unaltered. In contrast to
IFN- and C5a/CB, stimulation with IL-3/IL-5/GM-CSF induced the
appearance of discrete clusters of CD63 that colocalized predominantly
with eosinophil MBP (Figure 1M-O). Interestingly, none of these
cytokines on their own at the same doses affected the CD63
localization. Translocation of CD63 during stimulation by IFN-,
C5a/CB, or IL-3/IL-5/GM-CSF was inhibited by the tyrosine kinase
inhibitor, genistein (10 6 M) (Figure 1P)
(n = 5).
Agonist-induced cotranslocation of CD63 and RANTES We have previously shown that IFN- stimulation of eosinophils
induces the rapid mobilization of RANTES to the cell periphery prior to
its release to extracellular space as an in vitro example of
PMD.11 To examine the association of CD63 and RANTES
translocation to cell periphery and selective release, double
immunofluorescent staining with specific antibodies to RANTES and CD63
was conducted on IFN--stimulated eosinophils. Following IFN-
stimulation (10 minutes) of eosinophils, immunoreactivity against
RANTES colocalized with that of CD63, with both signals translocating
to the periphery of the cells (Figure 1Q-S). The continued presence of
CD63 and RANTES crystalloid granules in the
cytoplasm of IFN--stimulated eosinophils was further evident by
differential interference contrast (DIC) imaging (Figure 1T). Mouse
IgG1 at equivalent concentrations was used instead of anti-CD63 and
anti-RANTES as isotype control (Figure 1L). In double immunostaining
procedures, appropriate isotype controls were run concurrently
(combination of anti-CD63/BODIPY-FL and mouse IgG1/TRITC or a
combination of mouse IgG1/BODIPY-FL and anti-MBP/TRITC); nonspecific
binding was not observed (Figure 2M-P).
Dexamethasone's effect on agonist-induced CD63 translocation To examine the effect of dexamethasone on agonist-induced intracellular translocation of CD63, freshly purified eosinophils were incubated in the presence of dexamethasone 106 for 60 minutes prior to agonist stimulation. Interestingly, dexamethasone inhibited the IFN-- (Figure 2A-D), C5a/CB- (Figure 2E-H), or IL-3/IL-5/GM-CSF- (Figure 2I-L) induced intracellular mobilization of
CD63 (n = 6).
CD63 surface expression in resting and stimulated eosinophils The surface expression of CD63 in freshly isolated eosinophils was examined by flow cytometry. Our results indicated that CD63 is expressed on the surface of resting eosinophils. Saponin-permeabilized cells showed a significant shift (50-fold) in mean fluorescent index (MFI), indicating a larger intracellular pool of CD63 (Figure 3) (n = 4). Total CD63 expression was similar in resting and agonist-stimulated cells, yet CD63 surface expression was enhanced after agonist stimulation (10 minutes), with C5a/CB inducing maximum up-regulation in contrast to the combination of IL-3/IL-5/GM-CSF, which induced a smaller degree of increased surface expression (Figure 4) (n = 4). The surface up-regulation of CD63 coincided with intracellular translocation of CD63 and RANTES upon agonist stimulation.
CD63 expression on enriched populations of crystalloid granules To understand the pattern of membrane fusion and enhanced CD63 expression on the eosinophil surface membrane, we studied the expression of CD63 on the surface of isolated crystalloid granules. We immunostained both a permeabilized and a nonpermeabilized population of dispersed and highly purified eosinophil crystalloid granules. The results indicate that CD63 was expressed on the surface of crystalloid granules. Interestingly, the MFI of permeabilized granules was increased approximately 2-fold in saponin-permeabilized granules, while the MFI for the isotype controls of permeabilized and nonpermeabilized populations of granules was the same (Figure 5) (n = 7). To ensure that granules were intact after isolation, granule preparations were immunostained with antibodies against MBP or RANTES, and no immunostaining was detected by flow cytometry against either of the 2 mediators in nonpermeabilized granule preparation. Only the permeabilized granules exhibited MBP and RANTES immunoreactivity (data not shown).
The association of CD63 translocation and enhanced surface expression with mediator release We examined the association between intracellular translocation and surface up-regulation of CD63 with mediator release upon agonist stimulation. We measured-hex release in the supernatant of
eosinophils stimulated with IFN-, C5a/CB, or IL-3/IL-5/GM-CSF (10 minutes). Our results indicate that agonist stimulation of eosinophils
(10 minutes) induced -hex release. C5a/CB induced maximum release in
contrast to the combination of IL-3/IL-5/GM-CSF, which induced minimum
release (n = 6). Release of -hex occurred concurrently with CD63
translocation to the cell periphery and the cell surface. Indeed,
-hex release triggered by various stimuli was associated with
agonist-induced enhancement of CD63 surface expression measured by flow
cytometry (Figures 6A and 4). In
particular, C5a/CB induced more -hex release in parallel with its
stronger effect on CD63 surface expression (Figures 6A and 4). We also measured RANTES and EPO in the supernatant of IFN--stimulated eosinophils. Our data indicate that IFN- stimulation of eosinophils (10 minutes) also induced the release of RANTES and EPO in a
dose-dependant fashion (Figures 6B and 6C, respectively). We examined
the effect of other agonists on CD63 translocation and surface membrane
up-regulation and tested tumor necrosis factor (10 ng/mL), phorbol
myristate acetate (10 ng/mL), and each of IL-3 (10 ng/mL), IL-5 (5 ng/mL), and GM-CSF (10 ng/mL) alone. The following combinations were
also tested: IL-3 and IL-5, IL-3 and GM-CSF, and IL-5 and GM-CSF.
Interestingly, none of the individual or combinations of agonists
listed induced CD63 translocation, surface membrane up-regulation, and
mediator release (data not shown).
The effect of glucocorticoids and tyrosine kinase inhibitor, genistein, on CD63 surface membrane up-regulation and mediator release The clinical efficacy of glucocorticoids in allergic inflammation is well documented. It is thought that steroids may partially mediate inhibition of the elaboration of proinflammatory and eosinophil-active cytokines.32 We therefore examined the potential inhibitory effects of glucocorticoids on-hex, EPO, and RANTES
release and correlated this with CD63 translocation and surface
membrane up-regulation. Treatment of eosinophils with dexamethasone (60 minutes) prior to agonist activation down-regulated -hex release
significantly (Figure 6A) and also inhibited agonist-induced CD63
membrane up-regulation (data not shown). Dexamethasone, in the absence
of agonist stimulation, did not alter the -hex spontaneous release
(Figure 6A). In addition, dexamethasone inhibited the IFN--induced
EPO and RANTES release. Dexamethasone had no effect on eosinophil
survival determined by trypan blue exclusion test.
To examine the potential involvement of tyrosine kinase activity in
CD63 translocation and cell surface up-regulation following agonist
stimulation, eosinophils were preincubated with genistein, a
broad-spectrum inhibitor of tyrosine kinases (n = 4). Genistein pretreatment of eosinophils down-regulated the IFN-
In this study, we have investigated the expression and subcellular
localization of CD63 in resting and activated human peripheral blood
eosinophils. We have further examined the association of CD63
intracellular localization and surface expression with selective mediator mobilization (RANTES) and release ( Piecemeal degranulation is a well-documented mode of mediator release in human eosinophils. In studies of individuals with asthma,33-35 allergic rhinitis,36,37 and nasal polyposis,13,38,39 approximately 67% of all airway mucosal eosinophils were shown to exhibit intact but lucent granules indicative of PMD. In allergen-exposed nasal airways, virtually all viable eosinophils in mucosal tissue showed signs of PMD under active disease conditions.36 Eosinophil granule alterations reflecting PMD have also been described in guinea pig models of asthma.40,41 In a previous study, our laboratory showed that selective release of
RANTES in eosinophils upon IFN- It is highly unlikely that the loss of CD63 immunoreactivity in the
internal population of crystalloid granules of stimulated eosinophils
is due to proteolytic degradation of CD63. First, unlike neutrophils or
mast cells, the eosinophil lacks potent proteases. Second, CD63 has
been shown to be resistant to protease activity due to its heavy
glycosylation on its extracellular-2 domain.44,45 Third,
while CD63 immunoreactivity was confined to the cell periphery after
IFN- Enhanced surface expression of CD63 in agonist-stimulated eosinophils
appeared to coincide with Surface up-regulation of CD63 in activated eosinophils appears to occur in vivo as well. Surface expression of CD63 was observed to be up-regulated in eosinophils recovered from bronchoalveolar lavage of allergic asthmatic patients.46 Our own studies showed that CD63 surface expression is enhanced in peripheral blood eosinophils from asthmatics (n = 5) compared with nonasthmatic individuals (n = 5) (data not shown). In localization studies, we provided evidence that CD63 is expressed on the membrane of purified crystalloid granules. Following permeabilization, granules showed 2-fold increase in immunoreactivity against CD63, potentially caused either by intragranular storage of CD63 or by the presence of granule membrane-associated CD63 on the inner leaflet of the granule membrane. However, it is unlikely that there will be an intragranular pool of CD63 approximately equal to the membrane-associated pool. Furthermore, a previous ultrastructural study of eosinophils localized CD63 to the membrane and not to the matrix of crystalloid granules.28 We therefore speculate that this 2-fold increase in immunoreactivity of permeabilized granules may be associated with the orientation of the epitopes whereby CD63 faces both directions, with half CD63 facing the inner leaflet of the granules. Potentially, this may be caused by the dynamic fusion of small (secretory and/or endocytic) vesicles with crystalloid granules. Indeed, a previous study showed that CD63 cycles between endocytic and secretory compartments in human endothelial cells.17 Another study also illustrated that members of the tetraspanin superfamily such as CD37, CD53, CD63, CD81, and CD82 are concentrated on the internal membrane of an MHC class II-enriched compartment,47 indicating that the "extracellular loops" of these tetraspanins may face the inner comportment of an intracellular organelle. Unlike IFN- Tyrosine kinase activity has been implicated in eosinophil activation
and mediator release.48-52 In our experiments, genistein fully inhibited agonist-induced (IFN- A number of studies have suggested a direct effect of glucocorticoids
on the survival, activation, adhesion, apoptosis, and degranulation of
eosinophils.53-57 In our hands, dexamethasone inhibited
agonist-induced intracellular translocation, mobilization, and cell
surface up-regulation of CD63. It also down-regulated agonist-induced
Members of the SNARE fusion complex have been proposed to play a central role in exocytosis.58 Our recent studies have shown that human peripheral blood eosinophils express members of the SNARE fusion complex, including VAMP-2,59 syntaxin-4,60 and SNAP-23.61 In addition to SNAREs, members of Rho-related GTPases, including Rac-2, have been identified in eosinophils and shown to be critical in the assembly of NADPH oxidase complex prior to the generation of superoxide.62 Yet, the potential interdependence between different elements of eosinophil exocytosis remains the subject of speculation. We are intrigued by the likely interplay between the CD63 and members of SNARE fusion complex and/or Rho-related GTPases and are currently pursuing studies to define the mode of these putative interactions. In conclusion, our novel observations on eosinophil CD63 have important implications for the expansion of current knowledge on the processes underlying eosinophil activation and exocytosis. Our data point to a new association of CD63 with agonist-induced eosinophil activation and mediator release, particularly PMD. These findings may ultimately lead to novel therapeutic strategies in the treatment of eosinophil-related allergic inflammatory diseases, particularly asthma.
The authors thank Drs Paige Lacy and Paul Forsythe, Department of Medicine, University of Alberta, for their helpful comments on the manuscript and Dr Harissios Vliagoftis for helpful discussion. We acknowledge Dorothy Putkowski, Department of Medical Microbiology and Immunology, University of Alberta, for her expert assistance in flow cytometry.
Submitted February 9, 2001; accepted January 24, 2002.
Supported by the Canadian Institutes for Health Research and the Alberta Heritage Foundation for Medical Research. R.M. is an Alberta Heritage Senior Medical Scholar. G.P.D. is the recipient of a Canada Research Chair in Respiration and is the current holder of the R. Fraser Elliott Chair in transplantation research from the Toronto General Hospital.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Redwan Moqbel, Director, Pulmonary Research Group, 550A Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada; e-mail: redwan.moqbel{at}ualberta.ca.
1. Gleich G-J, Adolphson C-R, Leiferman K-M. The biology of the eosinophilic leukocyte. Annu Rev Med. 1993;44:85-101[CrossRef][Medline] [Order article via Infotrieve]. 2. Wardlaw A-J, Moqbel R, Kay A-B. Eosinophils: biology and role in disease. Adv Immunol. 1995;60:151-266[Medline] [Order article via Infotrieve]. 3. Weller P. The immunobiology of eosinophils. N Engl J Med. 1991;324:1110-1118[Medline] [Order article via Infotrieve]. 4. Hamman K-J, Douglas I, Moqbel R. Eosinophil mediators. In: Busse WW,Holgate ST, eds. Asthma and Rhinitis. Vol 2. Oxford, United Kingdom: Blackwell Science; 2000:394-428. 5. Lacy P, Moqbel R. Eosinophil cytokines. Chem Immunol. 2000;76:134-155[Medline] [Order article via Infotrieve]. 6. Dvorak A-M, Weller P-F. Ultrastructural analysis of human eosinophils. Chem Immunol. 2000;76:1-28[Medline] [Order article via Infotrieve]. 7. Lacy P, Moqbel R. Eokines: synthesis, storage and release from human eosinophils. Mem Inst Oswaldo Cruz. 1997;92(suppl 2):125-133. 8. Ackerman S-J, Loegering D-A, Venge P, et al. Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin. J Immunol. 1983;131:2977-2982[Abstract].
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Top
Abstract
Introduction
namely, eosinophil
cationic protein, eosinophil peroxidase (EPO), and eosinophil-derived
neurotoxin
-granules of
platelets.
