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and Granulocyte-Macrophage Colony-Stimulating Factor1
*
Department of Pharmacology, School of Pharmacy, The Hebrew University-Hadassah Medical School, Jerusalem, Israel; and
David R. Bloom Center of Pharmacy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-neutralizing Abs. Most of the activity was heat
labile. TNF- was found to be predominantly (70%) responsible, while
histamine had no role. Mast cell sonicate also caused eosinophils to
release eosinophil peroxidase and to display morphologic signs of
activation. In conclusion, we have demonstrated that mast cells enhance
eosinophil survival in part through their activation to produce and
release the autocrine survival cytokine GM-CSF. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which is contained preformed, in mast cell granules (8), has
also been shown to slightly increase eosinophil viability (9). In
addition, mast cells can synthesize and release IL-3, IL-5, and
GM-CSF3 (10, 11), which cause
eosinophil growth and differentiation and keep them alive under in
vitro conditions, therefore being termed survival cytokines
(12, 13, 14, 15). Eosinophils are mature end cells that need to survive for extended periods of time in the inflamed tissues they have penetrated, to perform their function. Apoptosis, or programmed cell death and subsequent ingestion by macrophages, has been proposed as a possible mechanism that regulates eosinophil life span and clearance from tissues (16, 17, 18). Indeed, in vitro, the death of eosinophils induced by the absence of IL-3, IL-5, and GM-CSF is characterized by typical apoptotic features, including cell shrinkage and DNA fragmentation (16, 17, 19). Recently, as in other systems, it has been found that eosinophil apoptosis can be regulated by Fas ligand/Fas interactions (20, 21, 22). Inhibition of eosinophil apoptosis has been suggested as a key mechanism in the development of blood and more notably of tissue eosinophilia in asthma and other allergic disorders during the late and chronic phase of allergic reactions (23, 24, 25).
The aim of the present study was to analyze whether mast cells can influence eosinophil survival. To achieve this goal, human peripheral blood eosinophils were cultured in the presence of mast cell-derived products and their survival was assessed.
Evidence is presented in this study that mast cells are able to prolong
eosinophil survival partly by the induction of the autocrine production
of GM-CSF. One of the involved mast cell-derived mediators appears to
be TNF-. In addition, mast cells cause morphologic signs of
activation and induce EPO release from eosinophils. Our findings of the
influence of mast cells on eosinophil behavior seem to indicate a
central role for mast cells not only in initiating, but also in
perpetuating the allergic inflammatory response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cimetidine, compound 48/80, dexamethasone, dextran,
Ficoll-Paque, histamine dihydrochloride, metrizamide, pyrilamine,
toluidine blue, trypan blue, saponin, o-phenylenediamine
dihydrochloride, gelatin, heparin, and platelet-activating factor (PAF)
were purchased from Sigma (St. Louis, MO). ELISA kit for GM-CSF and for
TNF-; mouse-neutralizing mAbs for human GM-CSF, IL-3, IL-5, and CD5;
goat anti-rat TNF--neutralizing Abs; and apoptosis detection kit
(KNX 50-020) were purchased from R&D (Minneapolis, MN). rhGM-CSF and
rhTNF- were purchased from Genzyme (Cambridge, MA). Goat
anti-mouse Igs were purchased from Dako (Glostrup, Denmark).
Conjugated magnetic beads anti-CD16, anti-CD3, and
anti-CD14 were purchased from Miltenyi Biotec Gmbh (Bergisch
Gladbach, Germany). Tri-Reagent was purchased from Molecular Research
Center (Cincinnati, OH). Superscript Preamplification System for first
strand cDNA synthesis was purchased from Life Technologies
(Gaithersburg, MD). Taq, DNA polymerase, and dNTP mixture
were purchased from Boehringer Mannheim (Mannheim, Germany). GM-CSF
primers were purchased from Clontech Laboratories (Palo Alto, CA).
DMEM, RPMI 1640, FCS, tissue culture supplements, and trypsin were
purchased from Biologic Industries (Beit Haemek, Israel). 3T3 Swiss
Albino fibroblast cell line was obtained from American Type Culture
Collection (ATCC, Rockville, MD). Other reagents and chemicals were
obtained from Frutarom Chemicals (Haifa, Israel) and were of analytical
grade or the best grade available. Tissue culture plates and
plasticware were obtained from Corning (Corning, NY) and from Nunc
(Roskilde, Denmark).
Eosinophil purification
Eosinophils were purified, as previously described (26), from the peripheral blood of mildly atopic volunteers (eight males, four females, 16 to 48 yr old) who were not taking any oral treatment for their condition and whose blood eosinophilia ranged from 4 to 10%. Written informed consent was obtained from all of the volunteers according to the guidelines established by the Hadassah-Hebrew University Human Experimentation Helsinki Committee. Venous blood (50–150 ml) was collected in heparinized syringes. Blood was subjected to dextran sedimentation, and leukocytes were centrifuged on Ficoll-Paque (density = 1.077) for 25 min at 700 x g. Neutrophils in the granulocyte-enriched pellet were tagged with micromagnetic beads to anti-CD16 Abs. To eliminate any contamination of the eosinophils with mononuclear cells, anti-CD14- and anti-CD3-coated micromagnetic beads were also added to the anti-CD16/granulocyte mixture. By negative selection, highly purified CD16- eosinophils depleted of magnetically positive neutrophils (CD16+) and any contaminating mononuclear cells (CD14/CD3+) were obtained after passage of the granulocytes through a ferrous matrix column held in the field of a permanent magnet (MACS). Eosinophils were collected at a purity of 97 to 100%, as assessed by Kimura staining, and at a viability of 99%, as assessed by trypan blue staining.
Mast cell purification
Mast cells were isolated by a sterile procedure from the peritoneal cavity of Sabra rats, an outbred strain of Hebrew University (27). Rats underwent peritoneal lavage with Tyrode buffer containing 0.1% gelatin (TG buffer), and mast cells were purified on a metrizamide gradient (22.5%, >96% purity). To obtain mast cell sonicate, freshly isolated mast cells were resuspended in medium containing RPMI 1640, 200 µg/ml penicillin, 200 µg/ml streptomycin, 2 mM gentamicin, 2mM glutamine, 0.1 mM nonessential amino acids, and 5% v/v heat-inactivated FCS (enriched medium, EM), and were disrupted by bath sonication in ice for 1 min (Heat System Ultrasonics W 380, duty cycle 5 s, output power 50%). The sonicate was then microcentrifuged (5 min) at 4°C, and debris-free supernatants were aliquoted and stored at -70°C. For the preparation of supernatants, freshly isolated mast cells were resuspended in EM and were incubated at 37°C for 20 min with either compound 48/80 (3 µg/ml, activated cells) or EM alone (nonactivated cells). At the end of the incubation, mast cell viability was inspected by their ability to exclude trypan blue and found to be >98% in all of the samples. Suspensions were centrifuged (5 min, 150 x g), and supernatants were collected, aliquoted, and stored at -70°C; cell pellets were resuspended in EM, sonicated, and stored. To evaluate histamine released, a radioenzymatic assay was performed on cell sonicates and supernatants (28).
Eosinophil cultures
Freshly isolated eosinophils were seeded in 96-well plates at a concentration of 1 x 105/100 µl EM/well and cultured at 37°C in a humidified atmosphere of 5% CO2. To assess the effect of mast cell products on eosinophil survival (see below), either mast cell sonicate (1 x 105 mast cell/100 µl EM or otherwise stated concentration) or mast cell supernatants (1 x 105 mast cell/100 µl EM or otherwise stated concentration) were added to the wells immediately after the eosinophil seeding (time 0). Positive control cultures consisted of eosinophils incubated with rhGM-CSF (80 ng/ml). Cultures were thereafter incubated for different time points for up to 5 days. The various eosinophil parameters were evaluated at different time points, as described below.
In some experiments, eosinophil cultures were incubated with a 1:1 ratio of sonicate obtained from mouse 3T3 Swiss Albino fibroblasts or from human PBMC. PBMC were isolated from the same blood used to purify the eosinophils, as follows: the interface of the Ficoll-Paque gradient was recovered and washed twice by centrifugation in EM (5 min, 150 x g), after which the PBMC were resuspended in EM (4 x 106/ml) before sonication.
Experiments were conducted in triplicates.
Eosinophil survival assay
To determine the number of viable eosinophils in the different cultures described below, 20 µl of the cell suspension was removed from each well by gentle pipetting, on days 1 to 5 of culture. Viable and dead eosinophils were enumerated blindly after the addition of 20 µl of trypan blue in a hemocytometer. The number of viable cells is expressed relative to the number of cells seeded at the beginning of the culture and calculated as follows: percentage of viable eosinophils = (no. of trypan blue-negative cells on the day of test/no. of cells seeded on day 0) x 100.
Addition of neutralizing Abs against IL-3, IL-5, and GM-CSF to the eosinophil cultures and GM-CSF determination
To determine the possible contribution of IL-3, IL-5, or GM-CSF to the mast cell effect, mast cell sonicate (2.5 x 105/100 µl) was incubated with mouse anti-human-neutralizing Abs to the three different cytokines at three different concentrations (10 µg/ml, 1 µg/ml, 0.1 µg/ml) for 30 min at room temperature. The cross-reactivity of these Abs with rat cytokines is unknown. As a control, nonrelevant Ab, mouse anti-human CD5 was used. Thereafter, preincubated sonicate was diluted x2.5-fold and added to the eosinophil cultures to achieve a mast cell:eosinophil ratio of 1:1, and eosinophil viability was determined on day 3 of culture. In these experiments, to validate the neutralizing capacity of the anti-cytokine Abs, eosinophils were incubated with rhGM-CSF (0.5 ng/ml); rhIL-3 (1.25 ng/ml); and rhIL-5 (1.25 ng/ml) in the presence or absence of optimal neutralizing concentrations of respectively anti-GM-CSF (10 µg/ml), anti-IL-3, (3 µg/ml), and anti-IL-5 (3 µg/ml). Under these conditions, inhibition of eosinophil survival was 95, 90.5, and 97.5% (n = 2), respectively.
In another set of experiments to validate the eosinophils as the source of GM-CSF, dexamethasone (10-6 M) was added together with the mast cell sonicate to eosinophil cultures, and incubation was conducted for a period of 18 h. Thereafter, culture supernatants were recovered by centrifugation and were assessed for GM-CSF levels with a commercial ELISA kit according to the manufacturer’s instructions (R&D). The limit of assay sensitivity was 1 pg/ml. Results were expressed as pg/ml of GM-CSF generated by 1 x 106 eosinophils.
Morphologic analysis of eosinophil apoptosis and annexin V binding assay
To evaluate apoptosis by morphologic appearance, such as decreased cell size, nuclear condensation, and anucleation (16, 19), cytospins of eosinophils recovered from the different cultures (day 1 to 5) were prepared. The cytospins were then stained with Kimura staining (19), and evidence of apoptotic morphology was assessed by light microscopy in a blind fashion. The percentage of nonapoptotic eosinophils was calculated, as follows: percentage of nonapoptotic eosinophils = (no. of nonapoptotic cells on the day of test/no. of cells seeded on day 0) x 100.
An apoptosis detection kit (R&D) was used to quantitatively determine eosinophils undergoing apoptosis by virtue of their ability to bind annexin V and exclude propidium iodide (29).
For the assay, eosinophils (1 x 106/2 ml) were cultured with EM or EM containing mast cell sonicate at 1:1 ratio with the eosinophils, or EM containing GM-CSF (80 ng/ml) for 3 days in 24-well plates. Cells were processed and analyzed by FACS according to the manufacturer’s instructions.
RNA isolation and RT-PCR amplification
Total RNA was extracted from 3 to 5 x 106 (100% pure) freshly isolated eosinophils or eosinophils cultured for 18 h, either with mast cell sonicate (1:1 cell ratio) or EM alone in 12 cell culture plates by using the commercial reagent, Tri-Reagent, based on the acid guanidinium thiocyanate-RNA extraction technique (30).
First strand cDNA synthesis reaction was catalyzed by Super Script II RNase H-reverse transcriptase and oligo(dT)12–18 primer, according to the manufacturer’s instructions. The generated cDNA was amplified in a total volume of 25 µl using 1.25 U Taq DNA polymerase and 0.2 mM dNTP mixture, 0.4 µM GM-CSF primers, and 10% glycerol as a specificity enhancement. The primer sequences for GM-CSF were 5'-ATGTGGCTGCAGAGCCTGCTGC and 3'-CTGGCTCCCAGCAGTCAAAGGG, generating a fragment of 424 bp. The specificity of the primers was confirmed by the manufacturer. A DNA template for GM-CSF provided by the manufacturer was used as a positive control. The thermocycler PTC-100 (MJ Research, Watertown, MA) was used for the PCR amplifications with the following settings: 40 cycles at 94°C for 45 s, 65°C for 45 s, and 72°C for 2 min. At the end of the cycles, a primer extension period of 7 min at 72°C was included. Primers for ß-actin were used as a control to test the efficiency of cDNA synthesis. Amplified products were electrophoresed on 1.8% agarose gel stained with ethidium bromide and photographed under UV light.
Partial characterization of the mast cell-derived factor(s): heat
stability and addition of antihistamines or of anti-TNF- Abs
To assess whether the mast cell-derived factor(s) that influences eosinophil survival is heat stable, mast cell sonicate was incubated either at 56°C for 30 min or at 100°C for 10 min. At the end of the incubation, sonicates were microcentrifuged (7 min, 4°C) to get rid of possible precipitated material. The clear supernatants were aliquoted and stored at -70°C.
To test whether the effect of the mast cell sonicate on eosinophil survival is mediated by histamine, eosinophils were preincubated with the antihistamine drugs pyrilamine (anti-H-1) or cimetidine (anti-H-2) (10-4–10-6 M) for 30 min before mast cell sonicate was added to the cultures. In different wells, eosinophils were incubated with histamine dihydrochloride (10-5–10-8 M) in the absence of the mast cell sonicate. After 3 days, eosinophil survival was assessed.
To evaluate the contribution of TNF- to the mast cell-mediated
effect, the following experiments were conducted. TNF- was
quantified in the mast cell sonicate (107/ml EM) by a
commercial ELISA kit, according to the manufacturer’s instructions.
The limit of the assay sensitivity is 15 pg/ml. Results are expressed
as pg/ml of TNF- present in 107 mast cell sonicate. To
eosinophil cultures, either rhTNF- (0.004–4 µg/ml) or mast cell
sonicate that has been preincubated for 30 min with anti-rat
TNF--neutralizing Abs (0.0015–7.5 µg/ml) was added, and after 3
days, eosinophil viability was assessed.
Morphologic assessment of eosinophil activation
Eosinophils (1 x 105/100 µl) were incubated for 2 or 18 h with 100 µl of either EM, mast cell sonicate (cell ratio 1:1), or GM-CSF (80 ng/ml). At these two time points, cultures were examined under an Olympus BH-2-RFL-T2 inverted microscope. Cultures were photographed with a 200ASA Kodak film. The percentage of eosinophils with spindle-elongated shapes that correlates with cell adherence and activation was (31) assessed by the help of an eyepiece graduated grid in 300 eosinophils in 5 fields/well at a magnification of x400 by two independent observers in a blind fashion.
EPO determination
For EPO determination in the supernatants, eosinophils were cultured either in EM or in EM containing either mast cell sonicate (mast cell:eosinophil ratio 1:1 or 1:5, respectively) or PAF (10-6 M) for 20 min. EPO was assessed by an enzymatic-colorimetric assay (32). The substrate solution consisted of 0.1 mM o-phenylenediamine dihydrochloride in 0.05 M Tris buffer, pH 8, containing 0.1% Triton X-100 and 1 mM hydrogen peroxide. Aliquots (50 µl) of the sample were incubated with 50 µl substrate solution for 20 min at room temperature. The reaction was stopped by the addition of 100 µl of 4 M sulfuric acid, and the absorbance was determined at 490 nm by using a spectrophotometer.
Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis was performed by the Student’s paired t test. A p value of <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of mast cells on eosinophil survival was first assessed
by incubating human peripheral blood eosinophils with mast cell
sonicate at 1:1 cell ratio. In these experiments, eosinophil survival
was evaluated once per day for 5 days based on their ability to exclude
trypan blue. As shown in Figure 1
, the
mast cell sonicate increased and prolonged eosinophil survival. This
enhancing effect was already evident after 2 days and became highly
significant on days 3 and 4 of incubation. On day 3, 44 ± 0.6%
of the eosinophils cultured with mast cells were alive vs only
21.3 ± 0.8% in medium alone (n = 4). On
day 5, eosinophils cultured only in EM were nearly all trypan blue
positive, while 20% of the cells cultured in the presence of the mast
cell sonicate still excluded the dye.
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). At day
3 of culture, the presence of mast cell sonicate at eosinophil:mast
cell ratio of 5:1 and 2.5:1 significantly enhanced eosinophil survival,
which was measured by their ability to exclude trypan blue (27.2
± 0.5% vs 32.8 ± 0.3%, n = 6). At 1:1 ratio,
the mast cell sonicate increased eosinophil survival by 100%
(20.9 ± 0.7% vs 43.1 ± 0.6% eosinophils in EM vs
eosinophils with the mast cell sonicate, respectively,
n = 6). A further doubling in mast cell numbers
affected slightly, but not significantly, the eosinophil survival
(46.6 ± 0.6%). In these experiments, 87 ± 1.8% of
eosinophils incubated with an optimal concentration of GM-CSF were
alive on the third day of the experiments (n
= 4).
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Mast cell supernatants enhance eosinophil survival
To determine whether the mast cell-derived factor(s) that influences eosinophil survival and is contained preformed in the cells could be released upon mast cell activation, mast cells were incubated with compound 48/80 for 20 min.
Supernatants were collected from activated mast cells (71 ± 2.3%
histamine release, n = 4) and from control-EM incubated
mast cells (14 ± 1.7% histamine release) and added to the
eosinophils at cellular ratio of 1:1. After 3 days of incubation,
viable eosinophils were evaluated by their ability to exclude trypan
blue stain (Fig. 3). At this time point,
the percentage of viable eosinophils incubated with the supernatants of
activated mast cells was comparable with the percentage of viable
eosinophils incubated with mast cell sonicate (42.5 ± 0.5% vs
42.8 ± 0.8%, respectively, n = 3). This
indicates that the mast cell factor(s) can be released quickly upon
stimulation. Nonactivated mast cells spontaneously released the
factor(s), although to a much lesser degree than compound
48/80-challenged mast cells. In fact, 27.3 ± 1.3% of viable
eosinophils were present in cultures incubated with the supernatant of
nonactivated mast cells in comparison with 42.5 ± 0.5%
eosinophils incubated with the supernatant of activated mast cells
(n = 4, p < 0.0001). Incubation
of eosinophils with compound 48/80 alone did not influence their
survival (21.1 ± 1.4% in compound 48/80 vs 21.3 ± 1.4% in
EM, n = 4). The effect of mast cell supernatants on
eosinophil survival was concentration dependent (data not shown).
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In these experiments, we evaluated whether the prolonged survival
of eosinophils in the presence of mast cell sonicate is due to partial
inhibition of apoptosis that eosinophils undergo in the absence of
survival cytokines. Apoptotic and nonapoptotic morphology was evaluated
at the light microscope level after staining cytospin preparations of
eosinophils cultured with mast cell sonicate with Kimura staining. The
staining of eosinophils with either Kimura (19) or Wright-Giemsa
staining (16) makes it possible to distinguish among three populations
of eosinophils in culture: 1) normal looking fresh cells; 2) aging
cells, showing morphologic changes of apoptosis such as reduction of
cell size concomitant with nucleus and cytoplasm reduction; and 3)
apoptotic and necrotic anucleated cells that have been termed granule
bags. As shown in Figure 4, both at 3 and
4 days, the number of nonapoptotic eosinophils was significantly higher
in cultures containing mast cell sonicate in comparison with cultures
with medium alone (day 3, 42.5 ± 0.8% vs 14 ± 0.5%,
n = 4, p < 0.0001).
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Altogether, these experiments indicate that mast cell-induced eosinophil survival results from partial inhibition of the apoptotic process.
Role of survival cytokines on mast cell-induced enhancement of eosinophil survival
To evaluate the role of the survival cytokines (IL-3, IL-5, and
GM-CSF) in mast cell-induced enhancement of eosinophil survival,
eosinophils were incubated with mast cell sonicate that had been
preincubated with three different concentrations of either
anti-GM-CSF-, anti-IL-3-, or anti-IL-5-neutralizing Abs.
Anti-CD5 was used as a control, nonrelevant Ab. After 3 days,
eosinophil viability was evaluated by trypan blue assay. As shown in
Figure 5, only anti-GM-CSF Abs
significantly inhibited the mast cell sonicate-mediated effect. Similar
data were observed when anti-GM-CSF-neutralizing Abs were added
simultaneously with mast cell sonicate to the eosinophil cultures (not
shown). Anti-GM-CSF Abs were already highly inhibitory at the
concentration of 0.04 µg/ml (40.2 ± 2.3%, n =
6, p < 0.001) and reached a maximum of 61.7 ±
3.8% at the concentration of 0.4 µg/ml. At the same concentrations,
anti-IL-3 and anti-IL-5 did not influence the mast
cell-mediated effect. In addition, the percentages of viable
eosinophils in the presence of the highest concentration of either
experimental Ab alone or control Ab alone, in the absence of mast cell
sonicate, were similar (from 19.2 to 20%, n = 3) and
comparable with the one of eosinophil in medium alone (19.7 ±
0.9%, n = 3).
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To determine whether the cellular source of GM-CSF is the
eosinophils, the following two experimental approaches were taken.
First, eosinophils were incubated with mast cell sonicate in the
presence or absence of dexamethasone, which has been shown to inhibit
the production of GM-CSF in human blood cells and human fibroblasts
(33, 34). As seen in Figure 6, after
18 h in the presence of mast cell sonicate, eosinophils released
9.9 ± 4 pg/ml GM-CSF in culture supernatants. Addition of
dexamethasone (10-6 M), concomitantly with mast cell
sonicate to the eosinophils, decreased GM-CSF levels in the culture
medium to 2.6 ± 1 pg/ml (n = 3) comparable
with the levels found in eosinophil cultured in medium alone (2.1
± 0.6 pg/ml).
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A, while freshly isolated
eosinophils did not express GM-CSF mRNA (lane
1) after 18-h incubation with mast cell sonicate, cultured
eosinophils displayed a transcript for this cytokine (lane
2). We could not detect any transcript when eosinophils
were incubated in medium alone (lane 3). Both
freshly isolated and eosinophils cultured with mast cell sonicate or
medium alone expressed mRNA for ß-actin (Fig. 7B).
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Partial characterization of the mast cell factor(s) that induces
enhancement of eosinophil survival: heat stability, role of histamine
and of TNF-
To partially characterize the mast cell-derived factor(s) that increases eosinophil survival, its heat stability was evaluated by incubating eosinophils with mast cell sonicate after its heating at 56°C for 30 min or at 100°C for 10 min. Eosinophil viability on day 3 in EM was 21.2 ± 2.7%; in the presence of untreated sonicate was 38.3 ± 1.4%; in the presence of sonicate heated at 56°C was 26.3 ± 1.2% and of sonicate heated at 100°C was 26.5 ± 2.5% (n = 3). Therefore, both 56°C and 100°C treatments decreased the mast cell sonicate activity by 70.2 and 68.2%, respectively (p < 0.007 and p < 0.04).
Two major preformed mediators present in mast cells that are readily
released upon activation (35) and have been shown to influence
eosinophil properties (4, 9) are histamine and TNF-. Therefore, to
evaluate the role of histamine in mast cell-induced enhancement of
eosinophil survival, eosinophils were incubated with mast cell sonicate
in the presence of the antihistaminic drugs pyrilamine (anti-H-1)
or cimetidine (anti-H-2), or with increasing concentrations of
histamine, in the absence of mast cell sonicate. As shown in Table I,
neither pyrilamine nor cimetidine
could diminish the increased eosinophil survival induced by mast cell
sonicate (day 3 of culture). Moreover, histamine (Table II
) added to the culture medium of
eosinophils did not influence their viability, as shown by similar
percentages of viable eosinophils present in all of the different types
of cultures. The results of these experiments proved that the mast cell
effect on eosinophil viability is not mediated by histamine. To assess
the contribution of TNF- in our system, TNF- content in mast cell
sonicate was evaluated. TNF- was found to be contained in discrete
quantities in the mast cell sonicate (70 pg/107 cells,
n = 2). Next, the mast cell sonicate was preincubated
with different concentrations of anti-rat TNF--neutralizing Abs,
and then added to eosinophil cultures. As shown in Table III,
anti-TNF- Abs partially
inhibit the effects of mast cell sonicate. Maximal inhibition
(69.8 ± 5.1%) was obtained with an Ab concentration of 1.5
µg/ml (n = 4). These data would indicate that
TNF- is to some extent responsible for the mast cell survival effect
on eosinophils.
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To corroborate that mast cell-derived factor(s) activates eosinophils, we performed the following experiments.
Eosinophils were incubated with mast cell sonicate, medium alone, or
GM-CSF (80 ng/ml), and were observed under an inverted microscope for
morphologic evidence of cell activation (Fig. 8). After 2 h and 18 h in the
wells, in which eosinophils were cultured in medium alone, most cells
retained their spherical and refractile resting appearance and showed
little cellular adhesion. When eosinophils were incubated with mast
cell sonicate or GM-CSF, several of the cells became flat and assumed
an Amoeba-like shape at both of these points. These shape
changes have been described to be usually accompanied by an
exocytotic response (31).
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To further assess the capability of mast cell sonicate to induce eosinophil activation, the secretion of the preformed granular-stored mediator, EPO from eosinophils incubated with mast cell sonicate was evaluated. Mast cell sonicate at 1:1 cellular ratio released EPO from eosinophil after 20 min in a similar fashion to PAF, a potent eosinophil degranulator (0.136 ± 0.006 OD at 490 nM with mast cell sonicate vs 0.176 ± 0.010 OD with PAF, 10-6 M, n = 4). The mast cell-induced EPO release from eosinophils was significant when compared with EPO spontaneously released by eosinophils incubated with medium alone (0.132 ± 0.006 OD vs 0.078 ± 0.001 OD, n = 4, p < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was undertaken to investigate whether mast cells
could influence eosinophil survival, taking into account the proximity
of these cells in the inflamed tissues and the fact that mast cells are
the reservoir of pluripotent mediators, including histamine, TNF-,
and other cytokines that have been shown to influence eosinophil
behavior (4, 9, 43, 44).
To investigate the effect of mast cells on eosinophil viability, we incubated highly purified human peripheral blood eosinophils with rat peritoneal mast cell sonicate, as a source of mast cell preformed mediators. Eosinophil survival was evaluated by trypan blue exclusion test. Mast cell sonicate induced a dose-dependent increase in eosinophil survival. By day 3, 44% of the eosinophils cultured with mast cells were still viable, while only 21.3% survived in culture medium alone. The limited influence of the mast cell sonicate can be due to the interspecies differences between the mast cells and the eosinophils or to the fact that mast cells produce only a limited amount of the stimulatory molecule. In addition, it is possible that in the sonicate, together with survival-inducing mediators, inhibitory factors such as TGF-ß (45) are present.
We then assessed the specificity of the observed effect by incubating eosinophils with sonicate of human PBMC or of mouse 3T3 fibroblasts. PBMC sonicate did not affect eosinophil viability, demonstrating that these blood cells do not contain any preformed mediator that can affect eosinophil survival. In contrast, 3T3 fibroblasts sonicate, slightly but significantly increased eosinophil survival in the cultures. This result is related to previous data showing that coculturing eosinophils with 3T3 fibroblasts, although in the presence of GM-CSF, enhanced their in vitro survival (46). Interestingly, this enhancement of eosinophil viability was found to come in part from their integrin-mediated interaction with fibronectin, resulting in autocrine generation of survival cytokines (41).
Eosinophil-enhanced survival mediated by mast cells was due to partial inhibition of apoptosis, as assessed by morphologic changes and annexin V binding assay. When Kimura staining of eosinophils was used to recognize apoptotic cells by their classical shrinkage in volume, nuclear condensation or extrusion, and surface blebbing (19), it was found that the number of nonapoptotic eosinophils on days 3 and 4 was significantly higher in cultures with mast cell sonicate and comparable with trypan blue-negative cells, in comparison with eosinophils cultured in culture medium alone. In addition, while only 10.1% of eosinophils incubated in medium for 3 days were annexin V and propidium iodide negative, i.e., neither apoptotic or necrotic, 37.2% of eosinophils incubated with mast cell sonicate were nonapoptotic and alive at this time point, demonstrating that apoptosis was partially inhibited.
To determine whether the mast cell-derived survival factor(s) that is contained preformed in the cells could be released upon mast cell activation, mast cells were incubated for 20 min with the potent activator compound 48/80. The activation supernatants were then added to the eosinophils, and their viability was assessed. It was found that these supernatants were as potent as the mast cell sonicate in increasing eosinophil viability. This would demonstrate that the preformed mast cell factor(s) involved is released upon mast cell activation with a fast kinetics that would also exclude de novo protein synthesis.
Eosinophils have been shown to respond specifically to IL-3, GM-CSF,
and IL-5, and inclusion of these cytokines in the eosinophil-cultured
medium prolongs eosinophil life span by inhibiting apoptosis (39, 40).
Therefore, mast cell sonicate was added to eosinophils in the presence
of neutralizing Abs for the three cytokines. Interestingly, only
anti-GM-CSF Abs inhibited to a high degree (61.7%) the mast
cell-induced effect. Anti-IL-3 and anti-IL-5 Abs did not show any
inhibitory effect. It is possible, therefore, to conclude that the mast
cell effect is mediated at least in part by the survival cytokine
GM-CSF. We then contemplated what the cellular source of GM-CSF could
be. Eosinophils have been shown to express mRNA and immunoreactivity
for GM-CSF when activated with calcium ionophore, IFN-
, IL-3, or
IL-5 (47, 48). Moreover, we have shown recently that eosinophils
contain this survival cytokine preformed in their crystalloid granules
(49). On the other hand, to the best of our knowledge, it has not been
reported that rat peritoneal mast cells contain preformed GM-CSF. Thus,
to assess whether the eosinophils could be the source of GM-CSF, they
were incubated with mast cell sonicate alone or in the presence of
dexamethasone, which has been shown to inhibit GM-CSF production in
human blood cells and human fibroblasts (33, 34). After overnight
incubation of eosinophils with mast cell sonicate, GM-CSF was found in
discrete amounts in the culture supernatants, while its presence in the
cultures treated simultaneously with dexamethasone was drastically
reduced. This would indicate that mast cells have induced its
production by the eosinophils, and dexamethasone has inhibited this
process. To further confirm the autocrine production of GM-CSF by
eosinophils, RNA was extracted from eosinophils cultured overnight with
mast cell sonicate, and RT-PCR for GM-CSF was performed. Freshly
isolated eosinophils did not show a positive band for GM-CSF mRNA, but
following incubation with mast cell sonicate, cultured eosinophils
showed a strong signal for this cytokine. It is therefore conceivable
that mast cells increase eosinophil survival partially through
eosinophil activation to secrete and to de novo synthesize GM-CSF.
In a preliminary search for the mast cell-derived mediator(s)
responsible for the increased eosinophil survival, we performed heat
stability tests on the mast cell sonicate. Incubation of the sonicate
at 56°C or 100°C decreased their activity (
70%). Histamine is a
heat-stable molecule, while we found that rhTNF- partially loses to
some extent its survival activity toward eosinophils when heated (V.
Temkin and F. Levi-Schaffer, unpublished data). Histamine and TNF-
are two preformed mast cell mediators that can be quickly released
following activation (35). In addition, histamine has been shown to
stimulate eosinophil superoxide production (4), and together with
PGD2, to increase their cytosolic calcium (7). TNF-
can also activate eosinophil oxidative metabolism (43) and induces ECP
release (44). Therefore, to evaluate the possible contribution of
histamine in our system, eosinophils were incubated with mast cell
sonicate in the presence of either an anti-H-1 or anti-H-2
antagonist, or, in the absence of mast cells, with exogenous histamine.
Since antihistamine treatment of the mast cell sonicate did not
influence the eosinophil survival-enhancing activity, and histamine
alone was not able to mimic the mast cell-mediated effect, we excluded
histamine as the possible survival mediator. To evaluate the role of
TNF-, which we found in discrete quantity in our mast cell sonicate,
the sonicate was preincubated with neutralizing Abs for rat TNF-.
This treatment decreased by a maximum of 70% the survival-enhancing
activity of the mast cell, clearly indicating that TNF- is at least
partially responsible for the survival effect. Indeed, Valerius et al.
(9) found that rhTNF- increased eosinophil viability, although to a
lesser degree than rhGM-CSF. Moreover, we have also observed that
rhTNF- can enhance eosinophil survival, and that this effect is
partially blocked by neutralizing Abs to GM-CSF (V. Temkin and F.
Levi-Schaffer, unpublished data). Interestingly, it has been shown that
factors such as fibronectin, IFN-, LPS (41, 50, 51), and more
recently IL-13 (38) can all prolong eosinophil survival in vitro either
by induction of autocrine survival cytokine production by eosinophils
or by a direct effect. Since not all of the mast cell activity is
neutralized by anti-TNF- Abs, other mast cell-derived factors
should be identified that are responsible together with TNF- for the
described effect of eosinophil enhancement of viability.
From the next set of experiments, it became evident that mast cell
sonicate activates eosinophils also for secretion. This was
demonstrated first by morphologic evidence. Eosinophils incubated with
mast cell sonicate displayed signs of cell activation (31), such as
adherence and change in shape into an Amoeba-like flat shape
similar to that of eosinophils incubated with GM-CSF. The effect of
mast cell sonicate was optimal after 10 h, while the one of GM-CSF
was after 2 h. This can indicate that GM-CSF has a direct effect
on the eosinophils, while mast cells induce flat Amoeba-like
appearance of eosinophils in culture, possibly both by a direct effect
of a preformed medium and by an effect mediated by the autocrine
production of GM-CSF or other cytokines. Interestingly, it has been
shown that TNF- can induce morphologic changes in cultured
eosinophils similar to the ones we observed (43). We also found that
eosinophils incubated for 20 min with mast cell sonicate released
significant amounts of EPO in the culture medium.
In conclusion, the findings reported in this study provide strong evidence to indicate that mast cells are important physiologic triggers for eosinophil activation and survival. It is therefore conceivable that mast cells can interact in a synergistic fashion with eosinophils in the tissues to perform and perpetuate the allergic inflammatory response. Thus, it is now crucial to characterize all of the mediators and the mechanisms involved in these interactions because of the obvious importance of this interaction as a novel therapeutic target for allergic diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Francesca Levi-Schaffer, Department of Pharmacology, School of Pharmacy, The Hebrew University-Hadassah Medical School, 91120 Jerusalem, Israel. E-mail address:
3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage colony-stimulating factor; EM, enriched medium; EPO, eosinophil peroxidase; PAF, platelet-activating factor; rh, recombinant human.
Received for publication April 30, 1997. Accepted for publication January 29, 1998.
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