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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2508-2516
Intracellular Localization of Interleukin-6 in Eosinophils From
Atopic Asthmatics and Effects of Interferon 
By
Paige Lacy,
Francesca Levi-Schaffer,
Salahaddin Mahmudi-Azer,
Ben Bablitz,
Stacey C. Hagen,
Juan Velazquez,
A. Barry Kay, and
Redwan Moqbel
From the Pulmonary Research Group, Department of Medicine, University
of Alberta, Edmonton, Alberta, Canada; and Department of Allergy and
Clinical Immunology, National Heart and Lung Institute, London, UK.
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ABSTRACT |
Eosinophils, prominent cells in asthmatic inflammation, have been
shown to synthesize, store, and release an array of up to 18 cytokines
and growth factors, including interleukin-6 (IL-6). In this report, we
show that IL-6 immunofluorescence localizes to the matrix of the
crystalloid granule in peripheral blood eosinophils from atopic
asthmatics using confocal laser scanning microscopy (CLSM). Granule
localization of IL-6 was confirmed using dot-blot analysis and
enzyme-linked immunosorbent assay (ELISA) on subcellular fractions of
highly purified eosinophils produced from density centrifugation across
a 0% to 45% Nycodenz gradient. IL-6 was found to coelute with
eosinophil crystalloid granule marker proteins, including eosinophil
peroxidase (EPO), major basic protein (MBP), arylsulfatase B, and
 -hexosaminidase. Immunoreactivity to IL-6 colocalized with
granule-associated IL-2 and IL-5 in subfractionated eosinophils. We
also made the novel and compelling observation that interferon  (IFN), a Th1-type cytokine, stimulated an early elevation in
eosinophil IL-6 immunoreactivity. A 2.5-fold enhancement of IL-6
immunoreactivity in eosinophil granules was observed within 10 minutes
of IFN treatment (500 U/mL), as determined by subcellular fractionation and CLSM. These findings suggest that IFN has
short-term effects on human eosinophil function and imply that a
physiologic role exists for Th1-type cytokine modulation of Th2-type
responses in these cells.
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INTRODUCTION |
EOSINOPHILS, prominent cells in allergic
inflammation and asthma, have been shown to synthesize, store, and
release up to 18 inflammatory and regulatory cytokines and growth
factors,1 including interleukin-2 (IL-2), IL-4, IL-5, and
granulocyte/macrophage colony-stimulating factor
(GM-CSF).2-5 We have previously reported that human
asthmatic peripheral blood eosinophils express mRNA for
IL-6.6 In addition, IL-6-positive eosinophils have been detected in blood from normal donors, suggesting that IL-6 may be
constitutively synthesized and stored in unstimulated
eosinophils.7 The site of storage of IL-6 in eosinophils
could not be determined by immunocytochemical staining, although it
appeared to be associated with the crystalloid secretory granule,
because IL-6 immunoreactivity showed a granular pattern of
staining.6
IL-6 is a pleiotropic lymphokine shown to be released from a wide range
of tissue cell types, particularly fibroblasts, T cells, and peripheral
blood mononuclear cells.8,9 Its expression is usually
induced in cells by viral infection, lipopolysaccharide, or other
cytokines, depending on the cell type concerned. The biologic effects
of IL-6 range from stimulation of B-cell hybridoma and mouse
plasmacytoma growth (leading to enhanced monoclonal antibody
production), B-cell terminal differentiation, T-cell proliferation, and
induction of cytotoxicity, to proliferation of hematopoietic progenitor
cells.8,9 Many actions of IL-6 occur in synergy with other
cytokines, principally IL-1, IL-3, and GM-CSF. IL-6 has been shown to
be a cofactor that potentiates IgE production from switched B cells by
enhancing the effects of IL-4,10 suggesting a role for IL-6
in atopy and Th2-type responses. Recent evidence suggests that IL-6 may
be involved in the pathophysiology of bronchial
asthma.11,12 Elevated levels of circulating IL-6 were
observed in asthmatic subjects (both symptomatic and asymptomatic)
compared with normal controls. IL-6 levels were further increased
during natural exacerbation of asthma compared with asymptomatic
periods.11 Furthermore, bronchoalveolar lavage levels of
IL-6 were found to increase after occupational allergen
challenge.12
We have previously shown that mRNA encoding IL-6 and the released
product (by in situ hybridization and enzyme-linked immunosorbent assay
[ELISA], respectively) were upregulated after treatment of highly
purified human asthmatic eosinophils with interferon (IFN).6 The effect of IFN on stimulating the release
of other eosinophil-derived cytokines and chemokines has also been observed.13-15 IFN is a cytokine described as the
prominent product of Th1-type lymphocytes in both mouse and
human,16,17 and is usually associated with a wide range of
bacterial and viral infections. Serum levels of IFN have been shown
to be elevated in acute severe asthma.18 Previous studies
have demonstrated that IFN can influence human eosinophil
cytotoxicity and receptor expression following long-term (>12 hours)
stimulation.19,20 The observation that IFN, a Th1-type
cytokine, may be able to stimulate the release of a Th2-type cytokine
(IL-6) from human eosinophils suggests that a shift is needed in our
current appreciation of the relationship between these two types of
immune responses, at least at the level of the eosinophil.
The aim of the present study is to determine the intracellular site of
storage for eosinophil-derived IL-6, and to analyze the possible
effects of IFN on IL-6 mobilization within the eosinophil. We
hypothesized that (1) IL-6 is associated with the matrix of the
secretory granules in human eosinophils, and (2) IL-6 storage and
release in human eosinophils is regulated by IFN in a time-dependent manner. We tested these hypotheses using a combination of in vitro assays, confocal laser scanning microscopy (CLSM), and subcellular fractionation on peripheral blood eosinophils obtained from atopic asthmatic subjects. We examined the colocalization of IL-6 with known
granule proteins in the presence or absence of IFN stimulation. Our
results suggest an early effect of IFN on eosinophil-derived IL-6
storage and mobilization within the cell.
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MATERIALS AND METHODS |
Materials.
Di-isopropyl fluorophosphate (DFP), phenylmethylsulfonyl fluoride
(PMSF), leupeptin, aprotinin,
N -p-tosyl-L-arginine methyl ester
(TAME), 4-methylumbelliferyl sulfate, 4-methylumbelliferyl N-acetyl--D-glucosaminide, -nicotinamide
adenine dinucleotide (reduced form), Coomassie Blue G, and sodium
pyruvate were purchased from Sigma (Poole, Dorset, UK). Adenosine
triphosphate (ATP) was obtained from Boehringer (Mannheim, Germany).
Nycodenz was purchased from Nycomed Pharma (Oslo, Norway) and from
GIBCO-BRL Life Technologies (Grand Island, NY).
Preparation of eosinophils.
A sample of peripheral blood (100 mL) was obtained from mild atopic
asthmatic subjects displaying eosinophilia more than 10% and who were
not receiving oral corticosteroids. Red blood cells were removed by
dextran sedimentation, with the remaining cells subjected to density
centrifugation on Ficoll to obtain a granulocyte pellet. Eosinophils
were then purified by immunomagnetic selection using the MACS system
(Becton Dickinson, Cowley, UK). This method uses the expression of CD16
antigen on neutrophils, because this antigen is absent from resting
eosinophils, as previously described.2,3,5,6,20,21 Briefly,
anti-CD16 monoclonal antibody (MoAb) bound to micromagnetic beads
(Miltenyi Biotec, Bergisch-Gladbach, Germany) was incubated with the
granulocyte pellet for 40 minutes at 4°C. Contamination by
mononuclear cells was avoided by coincubating anti-CD14- and anti-CD3-coated micromagnetic beads (Lab Impex, Teddington, Middlesex, UK; and Miltenyi Biotec). The mixture was then passed through a ferrous
matrix column held in the field of a permanent magnet. Highly purified
CD16 eosinophils (>99%) were obtained by negative
selection, depleted of the immunomagnetically positive neutrophils
(CD16+).
Subcellular fractionation.
Eosinophils were subjected to subcellular fractionation as previously
described.2,5 Briefly, at least 5 × 107
purified eosinophils were treated with 2 mmol/L DFP, a serine protease
inhibitor, for 5 minutes at room temperature before sedimenting at
240g for 5 minutes. The pellet was resuspended in
ice-cold 0.25 mol/L HEPES-buffered sucrose (containing 10 mmol/L HEPES, 1 mmol/L EGTA, and 5 µg/mL each of leupeptin, aprotinin, and TAME, pH
7.4), and the cells were centrifuged again at 4°C. Cells were resuspended in homogenization buffer (HEPES-buffered sucrose
supplemented with 2 mmol/L MgCl2 and 1 mmol/L ATP) to
optimal subfractionating density, between 10 and 15 × 106/mL, and subjected to 10 to 15 passes through a
ballbearing homogenizer (EMBL, Heidelberg, Germany) possessing 11 µm
clearance. 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, High Wycombe, UK).
The postnuclear supernatant was subjected to equilibrium density
centrifugation at 100,000g for 1 hour at 4°C. Twenty-four
0.4-mL fractions were collected from each preparation and stored at
4°C no longer than overnight or  80°C until used. The density of
each fraction was calculated from its respective refractive index.
Marker enzyme assays.
A total of four marker enzyme assays were used for locating specific
subcellular organelles to fractions collected from density gradient
centrifugation. Arylsulfatase B and -hexosaminidase activities were
measured in each fraction as markers for secretory granules and
lysosomes, using the method described by Levi-Schaffer et
al.5 Briefly, 50 µL diluted fraction (1:10 in
HEPES-buffered sucrose) was added to a black 96-well microplate and
mixed with 50 µL arylsulfatase B substrate solution (10 mmol/L
4-methylumbelliferyl sulfate in 0.2 mol/L acetate buffer, 6 mmol/L lead
acetate, and 0.1% Triton X-100, pH 5.6) or 50 µL -hexosaminidase
substrate solution (1 mmol/L 4-methylumbelliferyl
N-acetyl--D-glucosaminide in 0.2 mol/L citrate
buffer and 0.1% Triton X-100, pH 4.5) before incubating at 37°C for
1 hour. The reaction was terminated by addition of 150 µL ice-cold
0.2 mol/L Tris, and the fluorescence was measured in a Titertek
Fluoroskan (Huntsville, AL) microplate reader (excitation 340 nm and
emission 450 nm). For marking the presence of secretory
granules, we measured eosinophil peroxidase (EPO) activity adapted from
White et al22 for microtiter plates, and in later
experiments, tetramethylbenzidine substrate solution ([TMB] Sigma,
Oakville, Ontario, Canada) was used as a safer substrate for EPO in place of the more commonly used o-phenylenediamine HCl. Cytosolic activity was detected by assay of lactate dehydrogenase (LDH) as previously described.5 Plasma membrane activity
was assessed by the dot-blot method with antibody to CD9 as previously described.5 Protein measurements were made using the
Bradford dye-binding protein assay, with bovine serum albumin as the
standard.
Cytokine ELISA.
IL-6 and IL-2 were assayed in fractions using Quantikine ELISA kits
(British Biotechnology, Oxford, England; and R & D Systems, Minneapolis, MN). These assays have a detection sensitivity of 0.08 and
6 pg/mL, respectively. Assays were performed in duplicates (whenever
possible) of undiluted fractionated material. IL-5 was quantified using
an anti-human IL-5 MoAb as previously described.23 The
detection sensitivity of the assay was 6.25 pg/mL.
Dot-blot analysis.
This technique was used to confirm the presence of IL-6 shown by ELISA
and to detect the granule-associated proteins, major basic protein
(MBP) and eosinophil cationic protein (ECP), and the eosinophil plasma
membrane marker CD9. For detection of IL-6, an anti-human IL-6 MoAb
was used in parallel with a rabbit anti-mouse antibody used as a
negative control (British Biotechnology). MBP was detected by an
in-house mouse MoAb BMK-13 (supernatant IgG1) that has been carefully
validated, whereas ECP was detected using EG2 MoAb (supernatant IgG1;
Pharmacia, Uppsala, Sweden). Anti-CD9 MoAb (purified IgG1) was
purchased from Becton Dickinson. Both anti-CD3 MoAb (supernatant IgG1)
and anti-CD5 MoAb (purified IgG1) were used as irrelevant negative
controls for these MoAbs (Becton Dickinson). A total of 2 µL of the
supernatants were placed on nitrocellulose strips, allowed to dry, and
blocked in 5% milk powder (Sigma). The blocked membrane strips were
incubated with appropriate antibodies, and after extensive washings in
PBS-Tween 20, they were incubated with biotinylated anti-mouse or
anti-goat antibody followed by streptavidin-alkaline phosphatase,
washed, and developed. In the IL-6 dot-blot assay, recombinant IL-6 was used as a positive control. Fractional activities of the markers were
assessed by staining density, assigned arbitrary units, and converted
to the percentage of total activity for each specific protein.
Double labeling and CLSM.
Cytospins of eosinophils (100 µL 0.5 × 106 cells/mL
in RPMI supplemented with 20% FCS) were made by vortexing slides in a
Cytospin 2 (Shandon, UK) centrifuge (800 rpm for 2 minutes) followed by fixation in 2% paraformaldehyde in PBS for 10 minutes. Slides were
subjected to a wash step (five washes in Tris-buffered saline [TBS]),
followed by incubation in blocking solution (2% human IgG [Sigma
Reagent Grade] in H2O) in a humidified container for 1 hour. After a second wash step, TBS containing 1% rat anti-human IL-6
MoAb (50 µg/mL) labeled with FITC (Pharmingen, San Diego, CA) was
added, and the slides were incubated for 1 hour. Slides were subjected
to a wash step again before adding 1% BMK-13 in TBS and incubating for
1 hour. Bound BMK-13 antibody was detected by addition of 1% (50 µg/mL) Texas Red-labeled goat anti-mouse antibody (Pharmingen) to
washed slides and incubation for 1 hour. For comparison, 1%
FITC-labeled rat IgG2a was used as an isotype control (Pharmingen).
After a final wash step, 10 µL antibleaching agent (0.4%
n-propyl gallate [Sigma] in 3:1 glycerol:TBS) was dropped
onto each slide before cover slip attachment. Slides were examined
using a 100× objective under a Leica confocal laser scanning microscope (Heidelberg, Germany). This instrument contained a krypton-argon laser to allow simultaneous scanning of two excitation wavelengths (488 and 568 nm) so that two images could be acquired from
a single pass to minimize bleaching of the slides. Differences between
the photomultiplier tube sensitivities of the two fluorochrome emission
spectra were compensated during collection of the data to obtain images
of equivalent brightness. Images were stored on computer and
transferred to Adobe Photoshop (Adobe Systems Inc, Mountain View, CA)
for cropping and sizing.
Data presentation.
The bioactivity of eosinophil granule, membrane, and cytosol
constituents after fractionation, including IL-6 quantitation by ELISA
(picograms per milliliter) and dot blot, is expressed as the frequency
distribution as previously described,5 except for the time
course of IFN stimulation, where IL-6 is quantitatively displayed as
picograms per fraction.
| |
RESULTS |
Immunocytochemistry using APAAP.
Highly purified eosinophils from asthmatic subjects were examined using
the APAAP staining technique for the presence of IL-6 immunoreactivity.
Anti-IL-6 binding revealed cytoplasmic and/or granular
staining of IL-6 in a subpopulation of cells (25% to 50%; Fig
1a), suggesting that IL-6 may be stored
inside the secretory granules, in the same manner as previously
observed for other eosinophil-derived cytokines.2,3,5
Staining of eosinophils with an isotype control antibody was negative
(Fig 1b).
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| Fig 1.
Photomicrograph of a human eosinophil detected in a buffy
coat cytospin. (a) The eosinophil was stained specifically with mouse
monoclonal anti-human IL-6 using the APAAP technique, showing a
granular pattern of immunoreactivity as compared with the negative isotype control (b) (original magnification ×100).
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Subcellular fractionation.
Using subcellular fractionation of eosinophils, we successfully
separated some of the organelles in these cells, allowing analysis of
organelle-specific protein expression. The secretory granules, plasma
membrane, and cytosol were clearly resolved after density gradient
centrifugation (Fig 2). Secretory granule
activity was detected by assays for EPO, ECP (Fig 3), and MBP, and was usually confined to a single peak. We have previously shown that the
pellet produced from pooled fractions corresponding to peak granule
protein activity contains enriched secretory granules.5
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| Fig 2.
Average of profiles (from 4 patients) of arylsulfatase B,
-hexosaminidase, protein, MBP, CD9, LDH, and EPO together with IL-6
immunoreactivity as determined by ELISA and dot-blot analysis. Measurements of activities were averaged and plotted as a function of
collected fractions.
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| Fig 3.
Four individual fractionations of human eosinophils in
which IL-6 immunoreactivity is compared with -hexosaminidase and EPO activities along with ECP immunoreactivity (using EG2 MoAb in dot
blot). (A to C) IL-6 measured by ELISA; (D) IL-6 measured by dot-blot
analysis.
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The plasma membrane and cytosol detected by peak anti-CD9 binding and
LDH activity, respectively, sedimented at densities much lower than
those for secretory granules. Immunoreactivity for the plasma membrane
antigen CD9 resolved into two distinct peaks. The first peak, present
in higher-density fractions, sedimented in fractions containing maximal
secretory granule activity. The second peak of CD9 immunoreactivity,
which was much larger than the first, appeared at the expected range of
densities (1.04 to 1.17 g/mL) for plasma membranes.24 The
protein assay showed enrichment of cellular protein in two peaks across
the gradient, corresponding to fractions containing secretory granule
activity and cytosolic activity. The cytosol, as identified by the
presence of LDH activity, was associated with fractions of a density
range of 1.03 to 1.07 g/mL, coeluting with the second peak of total protein.
-Hexosaminidase and arylsulfatase B activity, which coincided with
that of EPO, consistently overlapped and usually produced bisected
peaks, suggesting that at least two subpopulations of secretory
granules exist in eosinophils. Also displayed in Fig 2 is the profile
of IL-6 as measured by ELISA in a representative sample, which peaked
at or near the same density (~1.2 g/mL) as MBP and EPO.
The sedimentation profile of IL-6 immunoreactivity by ELISA and dot
blot, along with that of ECP, EPO, and -hexosaminidase, for four
separate fractionations are presented in Fig
3. In one of four preparations, IL-6
immunoreactivity could be resolved as a single well-defined peak.
Otherwise, two peaks of IL-6 immunoreactivity could be resolved on the
gradient in two preparations, whereas in the final preparation only a
shoulder on a single peak was apparent (Fig 3). These split peak
profiles overlapped with those of -hexosaminidase and arylsulfatase
B activities. In all cases, IL-6 immunoreactivity colocalized with
fractions possessing peak granule protein activity. Taken together,
these results suggest that IL-6 is associated with eosinophil secretory
granules, and may be present in more than one subpopulation of
granules. Using whole-cell samples, unstimulated eosinophils were found
to store an average of 25 ± 6 pg IL-6/106 cells
(n = 4). We have previously established that unstimulated eosinophils
are able to release an average of 190 ± 18 pg/mL IL-6 in
supernatants, which increased to 403 ± 214 pg/mL after 24 or 48 hours
of IFN stimulation (106 cells per sample).6
Dot-blot analysis was used to confirm the results of our ELISA for IL-6
(Fig 3D). All profiles of dot-blot analysis for IL-6 showed two peaks
of immunoreactivity, the first appearing in fractions containing
secretory granule activity and the second present in cytosolic
fractions as indicated by LDH activity. The most likely explanation for
the appearance of IL-6 immunoreactivity in the cytosolic fractions is
granule breakage during homogenization. In three of four cases,
dot-blot results correlated with those in ELISA determinations. The
fourth preparation of eosinophils yielded erratic values of IL-6
immunoreactivity by ELISA, although the dot-blot analysis for this
sample was similar to what was observed in the other three
preparations (Fig 3D).
Coelution of IL-2, IL-5, and IL-6 with granule markers.
In one preparation, IL-2, IL-5, and IL-6 immunoreactivities were all
determined by relevant specific ELISAs, and were detected in the same
fractions as those containing peak secretory granule activity (Fig
4). This finding supports previous results
showing IL-5 immunoreactivity in the secretory granules of
eosinophils.25
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| Fig 4.
IL-2, IL-5, and IL-6 immunoreactivities in subcellular
fractions of eosinophils shown in comparison with arylsulfatase B, -hexosaminidase, and EPO activities. (A) IL-2, IL-5, and IL-6 measured by ELISA; (B) marker enzyme assays for secretory granules.
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CLSM.
In support of our findings using subcellular fractionation,
unstimulated eosinophils showed colocalization of IL-6 and MBP immunoreactivity to the crystalloid granules using CLSM in
double-labeled cells (Fig 5A to C). Yellow
regions in combined fluorescent imaging correspond to overlapping green
and red images, and suggest that the two stained proteins reside within
the same intracellular compartment. Isotype controls displayed minimal
fluorescence background after subtraction of autofluorescence (Fig 5D
and E). Interestingly, anti-IL-6 fluorescence produced characteristic
doughnut-like shapes corresponding to the granule matrix (Fig 5A, F, G,
and K). Upon increased magnification of combined images, these granular
shapes were found to possess red centers (crystallized MBP) surrounded by green fluorescence, corresponding to the core and matrix of the
crystalloid granules, respectively (Fig 5F). These observations suggest
that eosinophil IL-6 is stored within the matrix of the specific
secretory granule.
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| Fig 5.
Confocal microscopy of double-labeled eosinophils. (A to
C) Representative unstimulated eosinophils with the FITC channel corresponding to IL-6 (A), the Texas Red channel corresponding to MBP
(B), and the combined fluorescence (C). (D to E) Isotype controls for
FITC fluorescence and Texas Red fluorescence, respectively. (F)
Close-up of granules from an unstimulated cell showing doughnut-shaped IL-6 immunoreactivity surrounding red centers of MBP immunoreactivity. (G to J) Time course of IFN effects on IL-6 immunoreactivity in
eosinophils after (G) 10 minutes, (H) 6 hours, (I) 12 hours, and (J) 18 hours of stimulation by 500 U/mL IFN. (K to N) Same time course as G
to J, showing combined images for IL-6 and MBP (original magnification
×100).
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Effects of IFN stimulation.
The effect of IFN stimulation was examined in subfractionations of
unstimulated and stimulated eosinophils from the same donor at 10 minutes and 16 hours of IFN incubation. After 10 minutes of
stimulation by 500 U/mL IFN, immunoreactivity to IL-6 was elevated
over that of unstimulated cells (Fig 6),although it remained within the secretory granule fractions. The
granule-associated IL-6 was reduced to prestimulation levels after 16 hours of incubation with IFN.
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| Fig 6.
Eosinophils that were subfractionated to determine
effects of IFN incubation (500 U/mL) on IL-6 concentration or
localization. Quantification of IL-6 in each fraction was made by
ELISA. Marker enzyme assays used in these experiments were EPO and
-hexosaminidase. The experiments were conducted at different times
using purified blood eosinophils from the same donor. (A) Unstimulated
eosinophils, followed by eosinophils stimulated for (B) 10 minutes and
(C) 16 hours by IFN. An average of 50 × 106
eosinophils were used for each preparation.
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By confocal microscopy, 10 minutes of stimulation by IFN produced an
apparent intensification of anti-IL-6 fluorescence in the secretory
granules (Fig 5G). Anti-IL-6 fluorescence became diminished at 6 hours
of stimulation (Fig 5H) and dispersed throughout the cytoplasm after 18 hours of incubation (Fig 5J). By 24 hours of IFN stimulation, the
morphology of the cells deteriorated and staining became sporadic (data
not shown). During incubation with IFN, IL-6 immunoreactivity in
CLSM results became progressively dissociated from that of MBP in
combined images, suggesting that IL-6 had mobilized into separate
intracellular compartments (Fig 5K to N). However, we were unable to
confirm this finding in results from subfractionation, which may have
been a result of the very low levels of IL-6 in the individual
fractions.
| |
DISCUSSION |
In this study, we have shown for the first time that IL-6 is localized
to the matrix of secretory granules in eosinophils from atopic
asthmatics. We have based our findings on the comprehensive analysis of
subcellular fractions of eosinophils, correlation of cellular
components with IL-6 immunoreactivity, and fluorescent-labeled cells
using CLSM. The use of subcellular fractionation and CLSM combined
provides a powerful tool for determining the processes of storage and
intracellular trafficking of important proteins in inflammatory cells.
In subcellular fractionation, maximal IL-6 immunoreactivity was found
to coelute specifically with granule protein markers. We have
previously shown that fractions containing eosinophil granule proteins
are concentrated in crystalloid secretory granules.2,3,5
IL-6 immunofluorescence was found to localize to the matrix of the
crystalloid granule in CLSM analysis of cytospin preparations of human
eosinophils. Eosinophils are among the few inflammatory cells capable
of storing cytokines and chemokines in their secretory granules. These
stored products may be rapidly mobilized from intracellular sites of
storage and released locally during inflammation. Whether these
proteins are bioactive and exert their influence on the inflammatory
milieu in situ by paracrine, autocrine, or juxtacrine signaling remains
to be elucidated.
It is unlikely that IL-6 immunoreactivity detected in
granule-containing fractions results from internalized IL-6-bound
receptors, because secretory granules sediment at a much greater
buoyant density than anticipated for endosomal structures. These
structures would contain any endocytosed ligand-bound receptors, and
would be expected to sediment at a density equivalent to plasma
membrane.24 Moreover, earlier results have shown that
eosinophils transcribe and translate the gene encoding
IL-6,6 implying that IL-6 is synthesized within the
eosinophil.
The significance of the association of IL-6 immunoreactivity with
secretory granules lies in the possibility that IL-6 may be released by
regulated exocytosis from the eosinophil. Eosinophils have been shown
to undergo degranulation, measured by the release of -hexosaminidase
and by patch-clamp analysis, in response to intracellularly applied
agonists.26,27 So far, the mechanism of eosinophil-derived
cytokine release has not been investigated, although it is possible to
evoke secretion of IL-6 by IFN,6 suggesting that IL-6 is
released by receptor-mediated secretion.
We observed an early effect of IFN on the intensification of IL-6
immunoreactivity using two separate experimental approaches. This
finding compelled us to reevaluate our current appreciation of the
short-term effects of this cytokine. By subcellular fractionation of
eosinophils and CLSM, a 2.5-fold enhancement of IL-6 immunoreactivity was observed in eosinophil granules within 10 minutes of IFN treatment. Such short-term effects of IFN on IL-6 mobilization within eosinophils from atopic asthmatics may occur in patients with
acute severe asthma. These patients exhibit high levels of serum
IFN.18 Whether this is also relevant to viral
exacerbation of asthma remains to be established.28
The most plausible explanation for the observed short-term effects of
IFN on IL-6 immunoreactivity is that IFN may activate a
conformational change in the structure of IL-6 within the granules. Alternatively, IFN may stimulate the "unmasking" of putative prepro-IL-6, which is not recognized by the MoAbs used in this investigation. The specificity of the antibody for the prepro form of
IL-6 as opposed to the secreted form of this protein is not known. It
is unlikely that de novo IL-6 synthesis and vesicular trafficking into
the secretory granules would occur within such a short time to account
for the enhanced IL-6 immunoreactivity in response to IFN.
The CLSM results showing dissociation of IL-6 from MBP-positive granule
cores during prolonged stimulation by IFN suggest that some form of
translocation of IL-6 may occur between the secretory granules and the
plasma membrane, an observation that we were unable to confirm with
subcellular fractionation. However, the sensitivity of detection of
IL-6 immunoreactivity using CLSM is at least one order of magnitude
greater than that of ELISA measurement of IL-6 activity in subcellular
fractions. Based on CLSM results, extragranular anti-IL-6 staining may
be related to (1) newly synthesized IL-6 in the Golgi and its transport
to the secretory granules and/or (2) a smaller class of
secretory vesicles engaged in the process of piecemeal degranulation.
The latter process has been previously suggested for physiological release of eosinophil granule proteins.29 The biosynthetic
pathway of IL-6, as well as other cytokines, in eosinophils remains
unknown and is currently being investigated by our laboratory.
The putative role of eosinophil-derived IL-6 remains the subject of
speculation in asthma and allergic inflammation. IL-6 has been shown to
be associated with symptomatic and asymptomatic asthma and with natural
or induced exacerbations.11,12 The precise mechanisms
involving IL-6 in asthma are not clear, but it is likely that
eosinophils are an important source of this cytokine in asthmatic
responses. IL-6 is an important helper cytokine for primary
antigen-dependent T-cell activation and proliferation.30 Infiltration of CD4+ T cells has been shown to be an
important component of the inflammatory response in antigen-induced
late-phase allergic reactions in human skin,31
lung,32 and nose.33 IL-6 participates in
IL-4-dependent IgE synthesis from B cells10 and acts in
synergism with IL-3 and GM-CSF to enhance the proliferation and growth
of hematopoietic progenitors in humans.34,35 Furthermore,
IL-6 is involved in promoting secretion of IgA in mucosal tissue during
inflammatory reactions,30 and secretory IgA has been shown
to be a potent trigger of eosinophil degranulation.36 In
fibroblasts, IL-6 has been shown to inhibit cell growth,37
so the "repair" function of these cells may be influenced by IL-6
during allergic inflammation. IL-6 is released in association with
protective responses against infectious and parasitic
agents,9 and this may be related to the elevated eosinophil
numbers seen in helminth-induced infection.38 In summary,
eosinophils may serve as an important cellular source of IL-6 in
allergic and inflammatory reactions.
Eosinophils also store a number of other cytokines in their secretory
granules, such as IL-2, IL-4, IL-5, and
GM-CSF.2,3,5,25,39,40 Our results showing expression and
intracellular localization of IL-5 to the specific secretory granules
are in agreement with other reports describing detection of IL-2 and
IL-5 mRNA and their related products in eosinophils from patients with
atopic asthma.2,39-41
Storage of cytokines may lend eosinophils the potential to release
these preformed regulatory proteins rapidly and allow them to act
locally during inflammation. In addition, eosinophil-derived cytokines
may perpetuate inflammatory responses and prolong the survival of these
cells with damaging sequelae. Eosinophil synthesis and storage of
cytokines, chemokines, and growth factors in health and disease
requires further investigation to determine how these cells contribute
to the regulation of allergic inflammation.
| |
FOOTNOTES |
Submitted July 28, 1997;
accepted November 14, 1997.
Supported by The Wellcome Trust, UK; the Medical Research Council, UK;
Medical Research Council, Canada; and Alberta Heritage Foundation for
Medical Research.
Address reprint requests to Redwan Moqbel, PhD, Pulmonary Research
Group, 574 Heritage Medical Research Center, University of Alberta,
Edmonton, Alberta, Canada T6G 2S2.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We would like to thank Dr David Huston and Richard Dickason (Baylor
College of Medicine, Houston, TX) for assistance in measuring IL-5 in
one of our subfractionations; Professor Bastien Gomperts (University
College, London, UK) for invaluable advice, support, and supervision of
P.L. during the early phase of this research; and Matthew Wakelin and
Janet North (National Heart and Lung Institute, London, UK) for
technical assistance.
| |
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Abstract
Introduction
Abstract