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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 23-32
Rapid Mobilization of Intracellularly Stored RANTES in Response to
Interferon- in Human Eosinophils
By
Paige Lacy,
Salahaddin Mahmudi-Azer,
Ben Bablitz,
Stacey C. Hagen,
Juan R. Velazquez,
S.F. Paul Man, and
Redwan Moqbel
From the Pulmonary Research Group, Department of Medicine, University
of Alberta, Edmonton, Alberta, Canada.
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ABSTRACT |
The CC chemokine RANTES is synthesized, stored, and upregulated in
response to interferon- (IFN-) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic
asthmatics with 500 U/mL IFN- to analyze the kinetics of
mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay
(ELISA) to trace the movement of eosinophil-derived RANTES from
intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 ± 15 pg/mL
per 2 × 106 cells). RANTES appeared to be stored in at
least two intracellular compartments: the matrix of crystalloid
granules, detected by major basic protein and eosinophil peroxidase
activities, and a specialized small secretory vesicle present in light
membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN- stimulation. This effect was not observed in eosinophils treated with IFN- ,
interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating
factor (GM-CSF), or genistein followed by IFN-. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small
secretory vesicles.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
EOSINOPHIL ACCUMULATION is a hallmark of
allergic inflammation, particularly within the airway mucosa of
asthmatic subjects. Eosinophils are thought to be activated in response to local inflammatory stimuli by releasing an array of mediators. These
consist of cytotoxic granule proteins such as major basic protein
(MBP), eosinophil cationic protein (ECP), and eosinophil peroxidase
(EPO), products of respiratory burst, and lipid
mediators.1,2 In addition, eosinophils potentially
synthesize or produce up to 18 different cytokines and growth factors,
including interleukin-2 (IL-2),3,4 IL-4,5
IL-6,6,7 granulocyte-macrophage colony-stimulating factor
(GM-CSF),8-10 and RANTES.11-13 A number of
these proteins have been shown to exert autocrine effects on eosinophils, including RANTES.8,11,14
RANTES is a CC chemokine, which has been shown to be a potent
chemoattractant for CD4+/CD45RO+ T cells,
eosinophils, basophils, monocyte/macrophages, and mast cells.15-18 In addition to eosinophils, RANTES is
synthesized and/or released by a number of other cell types, such as T
cells, platelets, macrophages, endothelial cells, fibroblasts,
epithelial cells, and a mast cell line.16,19-23 Cutaneous
injection of RANTES was found to induce marked eosinophil recruitment
in human subjects, which was more rapid in allergic compared with
normal subjects.24 In earlier studies, RANTES has been
implicated in delayed-type hypersensitivity reactions19 and
in ongoing inflammatory processes in rheumatoid
arthritis.20 RANTES may have a role in contributing to the
infiltration of inflammatory cells in allergen-challenged airway
mucosal tissue in asthma. Although the expression and release of RANTES
in tissue and bronchoalveolar lavage (BAL) fluids from resting
asthmatic and normal subjects do not differ
significantly,25,26 the levels of RANTES were found to be
elevated in BAL fluids obtained from asthmatics 4 hours after allergen
challenge27 and in nasal fluids obtained from subjects with
allergic rhinitis after challenge with a grass pollen
extract.28 Increased RANTES secretion correlated strongly
with elevated tissue eosinophil numbers in both asthma and rhinitis.
Furthermore, RANTES has been shown to upregulate expression of
CD11/CD18 on monocytes29 and induce histamine release from
human basophils,30 suggesting that it may also have a role
in immediate-type allergic responses.
It has previously been shown that peripheral blood eosinophils express
mRNA for and release bioactive RANTES in response to serum-coated
beads, using immunocytochemistry (ICC), in situ hybridization (ISH),
and enzyme-linked immunosorbent assay (ELISA).11 A granular pattern of immunocytochemical staining of eosinophils was observed, suggesting that eosinophils store preformed RANTES in intracellular compartments. Interferon- (IFN-) was found to upregulate RANTES mRNA and protein expression in eosinophils after 16 hours of
stimulation.11 IFN- has been shown to be a viable
stimulus for eosinophils in a number of studies.6,7,9,31-33
We originally hypothesized that intracellular RANTES was secreted after
stimulation by IFN- in a time-dependent manner. However, preliminary
data indicated that IFN- had a more rapid effect on mobilization of
RANTES than previously anticipated. Thus, we propose that RANTES is
rapidly mobilized from intracellular stores in a piecemeal pattern of
degranulation. Eosinophils were purified from peripheral blood obtained
from atopic asthmatics and stimulated with recombinant human IFN-.
The release of RANTES was analyzed in cells using a combination of in
vitro assay, RANTES-specific ELISA, immunogold analysis, confocal laser
scanning microscopy (CLSM), and subcellular fractionation, and its
localization was compared with that of known crystalloid granule
proteins MBP and EPO. Our findings suggest that RANTES, a stored
product of eosinophils, is readily and selectively released in a
pattern akin to piecemeal degranulation after stimulation.
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MATERIALS AND METHODS |
Materials.
Mouse monoclonal alkaline phosphatase antialkaline phosphatase (APAAP)
detection kits were obtained from Dako (Glostrup, Denmark). Adenosine
triphosphate (ATP), aprotinin, N -p-tosyl-L-arginine methyl ester (TAME), phenylmethylsulfonyl fluoride (PMSF), Fast Red TR,
leupeptin, 4-methylumbelliferyl N-acetyl- -D-glucosaminide, -nicotinamide adenine dinucleotide (reduced form) (NADH), sodium pyruvate, and tetramethylbenzidine (TMB) substrate solution were purchased from Sigma (Oakville, Ontario, Canada). Genistein was obtained from Calbiochem Corp (San Diego, CA). Recombinant human IFN- was a kind gift from Dr Aziz Ghahary (Department of Surgery, University of Alberta). Nycodenz was purchased from GIBCO-BRL Life
Technologies, Ltd (Grand Island, NY). All reagents used in this study,
including media, were negative for lipopolysaccharides (LPS) activity, as determined using the E-Toxate assay (Sigma).
Preparation of eosinophils.
Peripheral blood (100 mL) was obtained from mild atopic asthmatic and
atopic nonasthmatic subjects displaying eosinophilia greater than 5%
and who were not receiving oral corticosteroids. After red blood cell
sedimentation in 5% dextran, remaining cells were subjected to density
centrifugation on Ficoll (Pharmacia Biotech, Uppsala, Sweden).
Eosinophils were then purified from the granulocyte pellet by
immunomagnetic selection using the MACS system (Miltenyi Biotec GmbH,
Bergisch Gladbach, Germany). Highly purified CD16
eosinophils (>99%) were obtained by negative selection, depleted of
neutrophils using anti-CD16-conjugated immunomagnetic beads as
described previously.7,10,34 Contamination by mononuclear cells and lymphocytes was avoided by coincubation with anti-CD14 and anti-CD3-coated micromagnetic beads (Miltenyi Biotec).
In vitro assay of granule protein release from eosinophils.
Purified human eosinophils were aliquoted at 2 × 106
cells/tube and treated with 500 U/mL IFN- at 37°C for various
times in RPMI-1640 (BioWhittaker, Walkersville, MD). The reaction was
terminated by placing tubes on ice and centrifuging cells at
400g for 5 minutes at 4°C. Assays were then performed for
RANTES immunoreactivity in supernatants using a Quantikine ELISA kit (R
& D Systems, Minneapolis, MN) with a detection sensitivity of 31.2 pg/mL. For detection of two other crystalloid granule proteins, EPO and
-hexosaminidase, assays for these were a modification of those
performed previously.4,7,10,35 Briefly, EPO activity was
assayed using TMB substrate solution by combining 50 µL sample with
150 µL substrate solution in a 96-well microplate and incubating at
room temperature for 15 minutes. The reaction was terminated by 50 µL
4 mol/L sulfuric acid and absorbance was read at 450 nm in a
spectrophotometric microplate reader (Vmax Kinetic Microplate Reader,
Molecular Devices, Sunnyvale, CA). For -hexosaminidase activity,
present in both secretory and lysosomal granules, 50 µL
sample was added to each well of a 96-well microplate and mixed with 50 µL substrate solution (1 mmol/L 4-methylumbelliferyl
N-acetyl--D-glucosaminide in 0.2 mol/L citrate buffer, pH
4.5, and 0.1% Triton X-100) before incubating (37°C, 1 to 2 hours). The reaction was terminated by the addition of 150 µL
ice-cold 0.2 mol/L Tris, and the fluorescence (excitation 360 nm,
emission 460 nm) measured in a Millipore CytoFluor 2350 plate reader
(Millipore, Nepean, Ontario, Canada).
Immunogold labeling and electron microscopy.
This procedure was a modification of a previously described
technique.5 Briefly, pelleted isolated eosinophils were
fixed in freshly prepared formaldehyde (2% in phosphate buffer [PB], 0.1 mol/L, pH 7.4) for 2 hours, embedded in Lowicryl K4M resin, and
subjected to an infiltration procedure involving a progressive lowering
of the temperature. Silver sections were cut and picked up onto
Formvar-coated copper grids. Before labeling, sections were blocked for
10 minutes with 0.14% glycine in PB. Additional blocking was performed
for 10 minutes in 3% horse serum. Grids were then floated on a
solution containing 20 µg/mL mouse monoclonal anti-human RANTES
antibody (R & D Systems) for 2 hours. A further blocking step
was performed using 0.14% glycine in PB. The immunoreactive label was
visualized by goat anti-mouse antibody conjugated to gold particles (20 nm diameter; E-Y Laboratories, Inc, San Mateo, CA) at 0.5 µg/mL in PB
for 2 hours. Sections were washed three times with 0.14% glycine in PB
before being rinsed for 3 minutes with distilled water. For
negative controls, we substituted anti-RANTES with mouse IgG1 as the
isotype control (20 µg/mL; R & D Systems). Staining with osmium
tetroxide was omitted to ensure visualization of gold particles in
electron-dense crystalloid granules in eosinophil sections.
Double-labeling and CLSM.
Cytospins of eosinophils (100 µL of 0.5 × 106
cells/mL in RPMI supplemented with 20% fetal calf serum [FCS]) were
made by spinning slides in a Cytospin 2 (Shandon Ltd, Astmoor, Runcorn,
UK) centrifuge (800 rpm for 2 minutes) followed by fixing in 2%
paraformaldehyde in phosphate-buffered saline (PBS) for 6 minutes.
These fixing and staining procedures were optimized as previously
reported36 and are satisfactory for visualization of
granule proteins in the absence of a permeabilization step. In our
hands, permeabilization agents were detrimental in obtaining optimal
cell morphology. Slides were then subjected to a wash step (five washes
in Tris-buffered saline [TBS], pH 7.4), followed by incubation in
blocking solution (2% bovine serum albumin [BSA]) in a humidified
container for 1 hour. Specific antibody binding was performed for 1 hour with TBS containing 5 µg/mL mouse monoclonal anti-human RANTES
antibody (R & D Systems). Immunoreactivity to RANTES was detected using 20 µg/mL BODIPY FL-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR) as previously optimized by our
laboratory.36 Slides were blocked again for 2 hours using
50 µg/mL goat anti-mouse IgG (Molecular Probes) and double-labeled
with 1% mouse monoclonal anti-human MBP antibody (BMK-13) in TBS for 1 hour. Bound BMK-13 was detected by incubating 5 µg/mL Texas
Red-labeled goat anti-mouse antibody for 1 hour (Caltag Laboratories,
San Francisco, CA). Mouse IgG1 (5 µg/mL) was used as an isotype
control (R & D Systems). After a final wash step, 10 µL of
antibleaching agent (0.4% n-propyl gallate [Sigma] in 3:1
glycerol:TBS) was applied to each slide before coverslip attachment.
Slides were examined using a 100× objective under a Leica CLSM
(Heidelberg, Germany). Images were collected and processed as
previously described.7
Subcellular fractionation.
Purified peripheral blood eosinophils were homogenized by repeated
passages through a ball-bearing cell homogenizer, with resulting
organelles separated by linear density gradient as described in earlier
reports.4,5,7,10 Briefly, at least 5 × 107 purified eosinophils from asthmatics were suspended in
ice-cold 0.25 mol/L HEPES-buffered sucrose (containing 10 mmol/L HEPES, 1 mmol/L EGTA, pH 7.4) and centrifuged at 4°C. Cells were
resuspended in homogenization buffer (HEPES-buffered sucrose described
above, supplemented with 100 µg/mL PMSF and 5 µg/mL each of
leupeptin, aprotinin, and TAME, 2 mmol/L MgCl2, and 1 mmol/L ATP) to 10 to 15 × 106/mL, and
subjected to 10 to 20 passes through a 12-µm clearance in a ball
bearing homogenizer (EMBL, Heidelberg, Germany). The homogenate was
centrifuged at 400g for 10 minutes, and the resulting postnuclear supernatant was layered onto an 8-mL linear Nycodenz gradient (0% to 45% Nycodenz dissolved in HEPES-buffered sucrose with
protease inhibitor cocktail) in a Beckman 14 × 89 mm Ultra-Clear centrifuge tube (Beckman, Palo Alto, CA). The gradient was subjected to
equilibrium density centrifugation at 100,000g for 1 hour at 4°C, and 24 × 0.4 mL fractions were collected from each
preparation and stored at  80°C until used.
Marker enzyme assays.
Three marker enzyme assays were used to obtain profiles of specific
subcellular organelles in fractions collected from density gradient
centrifugation. Activities of EPO and -hexosaminidase were
determined in supernatants and pellets using the same technique described earlier in this section. Cytosolic activity was determined using a modification of a microtiter plate endpoint assay37 for lactate dehydrogenase (LDH), where 10 µL of sample was mixed with
80 µL 1 mg/mL NADH and 0.75 mmol/L pyruvate in pH 7.5 phosphate buffer in a microtiter plate and incubated at 37°C for 30 minutes, followed by the addition of 80 µL 0.2 mg/mL
2,4-dinitrophenylhydrazine in 1 mol/L HCl and incubation at room
temperature for 20 minutes. The reaction was terminated by the addition
of 40 µL 3.5 mol/L NaOH and absorbance read at 450 nm. Plasma
membrane activity was determined by dot blot analysis (see below) with
monoclonal antibody (MoAb) to CD9 as previously
described.4,5,7,10
Dot-blot analysis.
Dot-blot analysis was performed to determine the distribution of CD9 in
subcellular fractions of resting and stimulated eosinophils. Anti-CD9
MoAb (purified IgG2a) was kindly provided by Dr A.R.E. Shaw (Cross
Cancer Institute, University of Alberta). A mouse monoclonal IgG2a
isotype control was used as the negative control for anti-CD9
(Pharmingen Canada, Mississauga, Ontario, Canada). Samples of each
fraction (2 µL) were pipetted onto nitrocellulose strips, allowed to
dry, and blocked in 5% milk powder. Blocked membrane strips were
incubated with 1:1,000 anti-CD9 antibody, and after extensive washings
in TBS, pH 7.6 + 0.2% Tween-20, were developed using APAAP staining
technique as previously described.10 The fractional
activities of anti-CD9 immunoreactivity were assessed by staining
density using a gel scanner, given arbitrary units and converted to
percentage of total activity in all fractions.
Data presentation.
The bioactivity of eosinophil granule, membrane, and cytosol
constituents after fractionation are expressed as frequency
distributions as previously described.10 RANTES was
quantitatively displayed as pg/fraction and pg/mL in fractions and
supernatants, respectively. Statistical comparisons were performed
using the Mann-Whitney test (one-tailed) and the Kruskal-Wallis one-way
analysis of variance. Results were considered significant when
P < .05.
| |
RESULTS |
Immunocytochemistry using APAAP.
In cytospin preparations of antibody-specific staining of buffy coat
from asthmatic subjects, RANTES immunoreactivity was detected in
eosinophils using APAAP staining. Morphologically identifiable
eosinophils, but not neutrophils or lymphocytes, displayed a mixture of
granular and extragranular staining corresponding to RANTES
immunoreactivity (Fig 1A). This indicates
that while RANTES appears to be stored in association with the unique
crystalloid granules of eosinophils, it may also be found in a number
of other intracellular compartments. The isotype control antibody
(mouse IgG1) was negative (Fig 1B).
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| Fig 1.
Human eosinophil detected in a buffy coat cytospin. (A)
The eosinophil was stained specifically with mouse monoclonal
anti-human RANTES (20 µg/mL) using APAAP staining, as indicated by
the arrow. Two unlabeled cells are also visible within this field, a
neutrophil possessing a multilobed nucleus (upper left) and a
lymphocyte (left of the stained eosinophil). (B) Isotype control using
mouse IgG1 antibody (20 µg/mL). Original magnification × 100.
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Release of RANTES after IFN- stimulation in vitro.
Incubation of eosinophils in culture media containing human recombinant
IFN- for prolonged periods (up to 24 hours) has been previously
demonstrated to upregulate the expression of a number of
eosinophil-derived cytokines, including RANTES.11 To
evaluate the time course of RANTES release during short periods of
stimulation by IFN- (500 U/mL), the amount of RANTES released by
stimulated cells was measured at 0, 10, 30, 60, 120, and 240 minutes (2 × 106 cells/time point). IFN- induced a rapid
release of RANTES from human eosinophils, reaching maximal levels after
60 to 120 minutes (four experiments). In a representative experiment,
eosinophils released an average of 80 ± 15 pg/mL RANTES after 120 minutes of IFN- stimulation (P < .05;
Fig 2A). The amount of maximal release
varied between the four donors (range, 74.5 to 302 pg/mL). After a
2-hour stimulation, the amount of RANTES detected in supernatants was
diminished to baseline values. Unstimulated eosinophils showed no
significant spontaneous release of RANTES (Fig 2A). Thus, IFN- was
found to induce rapid in vitro release of RANTES from human peripheral
blood eosinophils. In comparison, the release of EPO was assayed in the
same supernatants and plotted as a percentage of release induced by 60 minutes of incubation at 37°C with a maximally stimulating agonist,
serum-coated Sephadex (Pharmacia) beads, in a separate sample.
EPO release was potently induced by IFN- within 60 minutes of
incubation and diminished after 120 and 240 minutes of incubation.
However, because EPO activity is not always stable after its
release,38,39 we also measured -hexosaminidase activity
in these supernatants. -hexosaminidase activity continued to
increase in supernatants during continuous incubation, reaching values
exceeding the levels induced by serum-coated Sephadex beads, after 240 minutes of stimulation by IFN- (Fig 2B).
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| Fig 2.
Time course of RANTES, EPO, and -hexosaminidase
release from human peripheral blood eosinophils induced by 500 U/mL
recombinant human IFN-. (A) RANTES immunoreactivity in supernatants
of stimulated eosinophils (2 × 106 cells/time point) was
detected using a specific ELISA. Values represent averages of
triplicate measurements from cells at 0, 10, 30, 60, 120, and 240 minutes of incubation obtained from a representative donor. A similar
trend of release was observed in four separate donors. The dotted line
represents a single measurement of spontaneous release of RANTES from
eosinophils (duplicate values given for times 0 and 60 minutes). *
P < .05 compared with RANTES measured in supernatants at the
start of the time course by Kruskal-Wallis one-way analysis of
variance. The detection sensitivity of the RANTES ELISA was 31.2 pg/mL.
(B) IFN- also induced the release of eosinophil peroxidase and
another granule-stored product, -hexosaminidase, detected in the
same IFN--stimulated supernatants shown in (A). The amount
of granule protein release is expressed as a percentage of the release
induced by a maximally stimulating agonist (serum-coated Sephadex
beads) in another sample. Points and error bars represent the mean and
standard error of mean (SEM) of at least three
measurements.
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Immunogold labeling of RANTES.
Unstimulated eosinophils exhibited a granular staining pattern for
RANTES immunoreactivity, as indicated by the proximity of gold
particles to electron-dense granule cores
(Fig 3A and B). This pattern of
immunolabeling suggests that RANTES may be stored in association with
crystalloid granules. In addition, RANTES-specific staining was
distributed throughout the cell in the extragranular milieu (shown by
the arrowhead in Fig 3A). The isotype control showed negligible
background (Fig 3C).
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| Fig 3.
Transmission electron microscopy of immunogold-labeling
of RANTES in unstimulated eosinophils. (A) The arrow indicates gold
particles associated with electron-dense crystalloid granules, while
the arrowhead shows immunogold labeling of extragranular areas.
(B) Higher magnification of another cell. (C) Isotype control using
mouse IgG1 antibody. Original magnifications: (A), ×9,100; (B),
×34,000; and (C), ×6,900.
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CLSM.
To examine mobilization of stored RANTES using MBP as a marker for
eosinophil crystalloid granule, resting and IFN--stimulated eosinophils were subjected to immunofluorescent labeling with appropriate antibodies. Immunostained cells displayed discrete green
and red labels, which correspond to BODIPY FL-conjugated RANTES
(Fig 4A) and Texas Red-conjugated MBP (Fig
4B) immunoreactivity, respectively. Where colors overlapped in the
combined images, the immunofluorescence appeared yellow (Fig 4C),
suggesting that the two labeled proteins colocalize to the same
intracellular compartment. The isotype control exhibited negligible
immunoreactivity after subtraction of autofluorescence as previously
reported (data not shown36). At higher magnifications,
crystalloid granules appeared doughnut-shaped, with red centers
(crystalline core MBP) surrounded by green immunofluorescence to
RANTES, corresponding to the core and matrix of the crystalloid
granules, respectively (Fig 4D through F). There was only partial
overlap between MBP and RANTES immunoreactivities in the granules.
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| Fig 4.
CLSM of immunofluorescence staining of eosinophils. (A
through C) Unstimulated eosinophils labeled with BODIPY FL indicating
RANTES immunoreactivity (A), Texas Red corresponding to MBP (B), and
combined images (C). (D through F) Higher magnification of eosinophil
crystalloid granules showing matrix-associated doughnut-shaped RANTES
immunoreactivity (D), surrounding red-labeled cores of MBP
immunoreactivity (E), and combined images of the same structure (F). (G
through L) Combined images of RANTES and MBP, depicting time course of
IFN- (500 U/mL) stimulation, comparing (G) unstimulated cells with
those stimulated for 5 minutes (H), 10 minutes (I), 30 minutes (J), 60 minutes (K), and 16 hours (L). (M) Lower magnification of combined
images of eosinophils stimulated for 10 minutes with IFN-. (N)
Single-labeled unstimulated eosinophil, compared with (O) cell after 10 minutes stimulation (500 U/mL IFN-). (P) Inhibitory effect of
106 mol/L genistein added for 10 minutes before IFN-
stimulation. (Q) Cell incubated with 1,000 U/mL IFN- for 10 minutes.
Original magnification × 100 for all images, except for (M), ×63.
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In time-course experiments, eosinophils stimulated with 500 U/mL IFN- for 0, 5, 10, 30, 60 minutes, and 16 hours were fixed and stained for RANTES and MBP immunofluorescence. Interestingly, after
10 minutes of incubation with IFN-, RANTES immunofluorescence was
redistributed around the periphery of the cells (Fig 4I, M, and O),
while that of MBP remained within the core of the secretory granules.
The RANTES-specific immunofluorescence became visibly depleted in cells
after 1 hour of incubation with IFN-, although MBP activity was
still detectable (Fig 4K). After 16 hours of incubation, RANTES
immunoreactivity returned, indicating that some degree of replenishment
may have occurred (Fig 4L). These findings were reproduced in
eosinophils from three asthmatic donors, and images represent the
labeling pattern of the majority of cells in cytospin preparations.
To test the specificity of the IFN- response, eosinophils were
incubated with genistein (106 mol/L) for 10 minutes
before adding 500 U/mL IFN- for 10 minutes. Genistein is a broad
specificity tyrosine kinase inhibitor used to inhibit early steps in
the IFN- receptor signaling pathway after ligand
binding.40 It fully inhibited the effects of IFN- on
RANTES immunoreactivity in single-labeled cells (Fig 4P; a stimulated
single-labeled cell is shown for comparison in Fig 4O). Inhibition by
genistein was detected in greater than 90% of cells examined by CLSM.
In confirmation of this, we have also observed that genistein
(106 mol/L) inhibited RANTES release from
eosinophils in vitro (2 × 106) by 32% after 120 minutes of IFN- incubation (data not shown). In addition, we
incubated eosinophils with IFN- (1,000 U/mL) for 10 minutes and
found that it had no observable effect on the distribution of RANTES
immunoreactivity in eosinophils (Fig 4Q). We took advantage of the
small numbers of cells required for study by CLSM to examine the
effects of IFN- and genistein on eosinophils, as other in vitro
techniques require substantially larger numbers of cells. In a separate
assay, recombinant human IL-3 (25 ng/mL), IL-5 (10 ng/mL), and GM-CSF
(10 ng/mL) did not induce significant RANTES release from eosinophils
after 1 hour of stimulation (data not shown).
Subcellular fractionation.
Eosinophils (5 × 107) were homogenized, loaded onto
gradients of Nycodenz (0% to 45%) for ultracentrifugation, and
fractions containing intact organelles collected for later analysis by
ELISA and assays for enzyme activity. Intracellular compartments were identified in gradients by measuring marker enzyme activities within
individual fractions (Fig 5). Crystalloid
secretory granules were measured using assays for EPO and
-hexosaminidase, while plasma membrane activity was determined by
dot-blot analysis of the fractions using anti-CD9. Cytosolic fractions
were detected using an endpoint assay for LDH. Fractions with plasma
membrane activity are known to contain other light membranes including Golgi compartments, as determined by galactosyl transferase activity measured in subfractionated guinea pig eosinophils.41 We
have previously shown that the pellet produced from pooled fractions corresponding to peak granule protein activity, which sediment at high
buoyant densities typically observed for crystalloid granules, is
enriched in secretory granules.10
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| Fig 5.
Subcellular fractionation of unstimulated peripheral
blood eosinophils (5 × 107) obtained from an asthmatic
donor. Fractions were collected from a 0% to 45% linear Nycodenz
gradient and analyzed for marker enzyme activities to obtain profiles
of subcellular compartments. Marker assays used were eosinophil
peroxidase (secretory granules), -hexosaminidase (secretory granules
and lysosomal granules), CD9 (plasma membrane), and lactate
dehydrogenase (cytosol). Quantification of RANTES was performed by
ELISA for each fraction and is expressed as pg/fraction.
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In unstimulated eosinophils, RANTES immunoreactivity was detected in at
least two separate intracellular compartments (Figs 5 and
6A). The first peak of RANTES-specific
activity was detected in secretory granule-rich fractions, determined
by EPO and -hexosaminidase activity, while a larger peak was found
to be associated with the light membrane fractions, which overlapped
with CD9 immunoreactivity. Some CD9 immunoreactivity was visible in the
granule fractions, as described earlier,4,10,42 although
its optical density was too low to be detected, suggesting that a small
amount of CD9 is also intracellularly distributed in the eosinophil.
This observation confirmed our results from immunogold labeling and CLSM, which suggested that RANTES immunoreactivity only partially colocalized with secretory granules in unstimulated cells. Unstimulated eosinophils in this example were found to store approximately 72 pg
RANTES/106 cells.
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| Fig 6.
Subcellular fractionation of resting and
IFN--stimulated eosinophils (5 × 107 per
fractionation). These experiments were conducted at different times
using purified blood eosinophils from the same donor. Immunoreactivity
to RANTES was determined in individual fractions by ELISA and expressed
as pg/fraction. Profiles of EPO activity are shown here for comparison.
(A) Unstimulated eosinophils, followed by eosinophils stimulated for
(B) 10 minutes and (C) 60 minutes with 500 U/mL IFN-.
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Stimulation with IFN- (500 U/mL) induced a striking change in the
distribution of RANTES immunoreactivity in subfractionated eosinophils.
RANTES was rapidly depleted from light membrane-associated fractions
after 10 minutes of IFN- stimulation (Fig 6B), while some
immunoreactivity remained within the granule-associated fractions. Eosinophils stored approximately 23 pg/106 cells of
RANTES-specific activity after IFN- stimulation for 10 minutes, a
reduction of 68% compared with unstimulated cells. Moreover,
eosinophils stimulated for 60 minutes with IFN- showed a marked loss
of RANTES immunoreactivity from fractions containing peak secretory
granule activity (Fig 6C), which was translocated to plasma
membrane-associated fractions at 49% of the quantity measured in
unstimulated cells (35 pg RANTES/106 cells). These results
are in agreement with those of CLSM, in which eosinophils displayed
reduced RANTES activity after stimulation with IFN- (Fig 4J and K).
Each subfractionation profile was prepared on different occasions from
the same donor to allow comparison of control and stimulated cells.
IFN--stimulated eosinophils showed similar profiles of EPO activity
to that of unstimulated cells (Fig 6), although the peak of EPO
activity appeared to be partially diminished after 60 minutes of
IFN- stimulation.
| |
DISCUSSION |
We have shown for the first time that RANTES immunoreactivity in human
eosinophils is associated with the matrix of the crystalloid granules.
This is based on its close apposition to the crystalline core granule
protein marker, MBP. Interestingly, RANTES was also detected in an
extragranular compartment distinct from MBP- and EPO-containing
granules, which was readily released in response to IFN-. We propose
that the rapidly mobilizable RANTES is contained within a putative pool
of small secretory vesicles that is physically distinct from
crystalloid granule.
The profile of RANTES immunoreactivity in fractions from unstimulated
eosinophils suggests that the larger peak is likely to be associated
with small, light-density vesicles, which possess a greater buoyant
density than that of the plasma membrane, as indicated by CD9
immunoreactivity. It is important to note that these fractions do not
fully discriminate between endosomal membranes, Golgi, and plasma
membrane.41,43 The light membrane fractions are also likely
to contain the vesiculotubular structures previously described in
eosinophils.44
IFN- was observed to activate the release of RANTES from eosinophils
in parallel with two other granule-associated proteins, EPO and
-hexosaminidase. Invariably, the levels of RANTES in these
supernatants diminished to baseline values after 240 minutes of
incubation. Those of EPO were similarly found to become reduced after
120 minutes of incubation (Fig 2). These observations suggest that both RANTES and EPO may be sequestered by surfaces within the
assay after their release. Released EPO is likely to be lost to
surfaces due to its inherent highly cationic nature (isoelectric point
[pI] of 10.8).38,39 In addition, RANTES is rapidly
sequestered by cell surface glysoaminoglycans after its secretion,
which then foster the adhesion and activation of RANTES-responsive
cells.45 In our subfractionation studies, immunoreactivity
to RANTES was observed to shift to a very low-density peak after 60 minutes of stimulation with IFN-, which was shifted to the right in
comparison with the light-density peak of RANTES in unstimulated cells
(Fig 6A and C). This shift in the density of RANTES immunoreactivity suggests that released RANTES may be adhering to the glycosaminoglycans coating the surfaces of eosinophils. In support of the possibility that
specific eosinophil products may be lost to surfaces during in vitro
assay, the noncationic granule-derived enzyme -hexosaminidase (predicted pI of 5.4 to 5.9) was found to increase in supernatants during incubation with IFN- (Fig 2B).
Evidence for the existence of a putative small secretory vesicle in
eosinophils was provided in our studies on the effects of IFN- on
RANTES mobilization by CLSM. RANTES immunoreactivity appeared to be
transferred to the periphery of cells during IFN- stimulation,
apparently to a different vesicular compartment from the crystalloid
granules. Moreover, the subcellular fractionation profile of RANTES in
IFN--stimulated cells (after 10 minutes) showed that much of the
RANTES associated with light density fractions was depleted, while
granule-associated RANTES was maintained at a level equivalent to that
of unstimulated cells. The observation that IFN- exerted such a
rapid effect on mobilization and release of eosinophil-derived RANTES
was novel and compelling. This is complementary to an earlier report,
in which IFN- (1,000 U/mL) upregulated RANTES mRNA and protein
expression within eosinophils after 16 hours of
stimulation11 and provides further support for our recent
finding that IFN- rapidly elevated IL-6 immunoreactivity in human
peripheral blood eosinophils.7 After 60 minutes of stimulation by IFN-, nearly all of the detectable RANTES
immunoreactivity was colocalized with very light-density membranes as
determined by subcellular fractionation, indicating that the
crystalloid granule-associated RANTES may have been selectively removed
and transported via small secretory vesicles.
These observations suggest that eosinophils possess a unique mechanism
for selective, piecemeal release of mediators from the crystalloid
granules, probably through exocytosis of a population of small,
light-density vesicles. Such small secretory vesicles may be
responsible for shuttling crystalloid granule proteins from the
granules to the plasma membrane. Selective release of eosinophil
granule proteins has been described in earlier reports.46 A
similar pattern of piecemeal degranulation has been proposed based on
electron microscopy sections of eosinophilic degranulation, in
vivo.44 Previous studies have shown that eosinophils
undergo degranulation in response to intracellularly applied agonists, for example, guanosine 5'-0-(3-thiotriphosphate)
(GTPS),35,47 although the mechanisms regulating
exocytotic release have not yet been fully elucidated. We are currently
investigating the identity of the putative small secretory vesicles
with a view to determining their precise colocalization with known
eosinophil-derived intracellular proteins.
IFN- has been shown to stimulate eosinophils in vitro, as shown in
its ability to augment eosinophil-induced antibody-dependent cellular
cytotoxicity (ADCC)31 and induce the expression of FcRIII (CD16)32 and CD69.33 Further, IFN-
has been demonstrated to stimulate the release and/or upregulation of a
number of cytokines from eosinophils, including IL-3,48
IL-6,6,7 GM-CSF,8,9 and RANTES.11
Expression of functional IFN- receptors on human peripheral
eosinophils has been described recently.49 These and future
experiments continue to contribute to the intriguing observation that
IFN- can induce rapid changes in cytokine expression, receptor
upregulation, and mediator release in eosinophils.
Activation of RANTES mobilization and release from eosinophils by
IFN- was specific, as shown by genistein inhibition, a broadly
specific tyrosine kinase inhibitor, suggesting that IFN- acts on
these cells via the IFN- receptor, which activates the Jak-STAT
pathway.40 Furthermore, other cytokines such as IFN-, IL-3, IL-5, and GM-CSF were found to have little or no effect on RANTES
immunoreactivity or release into supernatants (Fig 4Q; data not shown).
The effects of IFN- were unlikely to be LPS-mediated because the
culture media containing IFN- used in this study, as well as all
other media, were negative for LPS as determined by routine E-Toxate
testing (data not shown).
All eosinophils tested in these experiments were derived from subjects
exhibiting atopy. It is possible that the effects of IFN- on RANTES
mobilization were due to enhanced susceptibility of the cells to
IFN- because of priming, for example.50 However, we have
also detected IFN--induced release of intracellularly stored RANTES
from eosinophils obtained from atopic nonasthmatic subjects. Thus, we
conclude that it is unlikely that the capacity of eosinophils to
generate a differential response to IFN- is dependent on the
asthmatic status of the donor.
In both human and murine studies, cytokines released during immune and
inflammatory reactions have been proposed to follow a dichotomy of Th1
and Th2-type responses depending on the nature of the stimulus
delivered to the immune system. Release of IFN- has been associated
with Th1-type cytokine responses in bacterial and viral infections
along with the suppression of atopy.51,52 On the other
hand, eosinophils are regarded as Th2-type effector cells with the
potential to respond to Th2-type cytokines thought to be associated
with the allergic phenotype.2,53,54 However, the
distinction between Th1 and Th2 cytokine profiles in most cases of
infection and inflammation in humans is less clearcut. Thus, while
IFN- potently inhibits granulocytic maturation and proliferation in
the bone marrow,55 the levels of IFN- have been found to
be increased in the sera of patients with acute severe asthma, who also
exhibit lung and tissue eosinophilia.56,57 The significance
of these findings may be that IFN- has a role in regulation of
eosinophil homeostasis by stimulating fully mature eosinophils locally,
while preventing excessive eosinophilia, as suggested by Valerius et
al.31 It is tempting to speculate that IFN- released
from virus-infected inflammatory and immune cells within the airway
mucosa in asthmatics may contribute to activation of resident airway
eosinophils during viral exacerbation of asthmatic
attacks.58
Eosinophil-derived RANTES is likely to play a role in paracrine,
autocrine, or juxtacrine signaling after its release, and its
bioactivity on other eosinophils, at least in vitro, has previously been demonstrated.11 Besides RANTES, eosinophils contain a
number of other cytokines in their secretory granules, such as IL-2, IL-4, IL-5, IL-6, tumor necrosis factor- (TNF-), and
GM-CSF.4,5,7,10,59,60 Storage of cytokines as preformed
mediators within secretory granules and their rapid release after
stimulation may lend eosinophils the potential to regulate local
inflammatory responses. Many of eosinophil-derived cytokines so far
described are produced in smaller proportions than those of other
immune cells, such as T cells. However, unlike eosinophils, T cells are
not known to have the capacity to store cytokines. Cytokines produced
from an overwhelming influx of actively degranulating eosinophils into the airways in asthma, for example, are likely to prolong their own
survival and perpetuate the inflammatory response. These exciting observations will be important in expanding our knowledge of the cytokine and chemokine network that regulates the processes of eosinophil activation and subsequent secretion of cytokines,
chemokines, and especially granule proteins with their recognized
damaging sequelae in allergic inflammation.
| |
ACKNOWLEDGMENT |
The authors thank Dr Vera Chlumecky (Department of Cell Biology and
Anatomy, University of Alberta), for her assistance in confocal laser
scanning microscopy; Richard Sherburne (Department of Medical
Microbiology and Immunology), for his help with electron microscopy; and Drs Klaus Erb and Harissis Vliagoftis for
their valuable comments on the manuscript. We are grateful to Drs
Andrew Shaw and Aziz Ghahary for their gifts of anti-CD9 and IFN-, respectively.
| |
FOOTNOTES |
Submitted October 19, 1998; accepted February 19, 1999.
Supported by the Medical Research Council, Canada, the University of
Alberta Hospital Foundation, the Alberta Heritage Foundation for
Medical Research, and the Glaxo Heritage Research Laboratory Award.
P.L. is a Parker B. Francis Fellow in Pulmonary Medicine and R.M. is an
Alberta Heritage Senior Medical Scholar.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Redwan Moqbel, PhD, FRCPath, Pulmonary
Research Group, 574 Heritage Medical Research Center, University of
Alberta, Edmonton, Alberta, Canada T6G 2S2; e-mail:
redwan.moqbel{at}ualberta.ca.
| |
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TOP
ABSTRACT
INTRODUCTION