Abstract
purpose. To examine the effect of human conjunctival fibroblasts on the survival
and functional activity of human peripheral blood eosinophils.
methods. Eosinophils were purified by negative immunoselection [magnetic
activated cell sorter (MACS), purity > 97%] from
volunteers with mild atopia. Fibroblasts were cultured from
conjunctival specimens of healthy donors. Eosinophils were cultured on
confluent monolayers of conjunctival fibroblasts or in culture medium
alone. Eosinophil survival was evaluated by the trypan blue exclusion
test. Eosinophil adherence was assessed by counting the attached cells
after washing the cultures. Eosinophil viability and adherence in
coculture were also assessed in the presence of
anti-granulocyte-macrophage colony-stimulating factor (GM-CSF),
anti-interleukin (IL)-3, and anti-IL-5 neutralizing antibodies.
Cocultured eosinophils were activated by lipopolysaccharide (LPS) after
4 days in culture, and eosinophil peroxidase (EPO) release was
determined as a marker of their activation.
results. Eosinophils cocultured with conjunctival fibroblasts had a
significantly increased viability of 35.9% (P = 0.004)
and 12.8% (P = 0.003) on days 4 and 8,
respectively. Fibroblast-conditioned medium did not enhance the
survival of eosinophils. The increase in eosinophil survival in
coculture was partially inhibited by anti-GM-CSF (P = 0.019), anti-IL-3 (P = 0.033), or anti-IL-5
(P = 0.011), whereas eosinophil adherence was
reduced by anti-GM-CSF alone (P = 0.034). LPS
activation of eosinophils cultured for 4 days with conjunctival
fibroblasts induced higher EPO release than in freshly isolated
eosinophils (P = 0.01).
conclusions. Human conjunctival fibroblasts induced prolonged survival and increased
secretory function of human peripheral blood eosinophils. Increased
survival is partially mediated by IL-3, IL-5, and GM-CSF. The coculture
of conjunctival fibroblasts with eosinophils can serve as an in vitro
system for the study of eosinophil behavior in the ocular surface and
of cellular interactions in allergic eye
diseases.
The ocular allergic inflammatory response involves a complex
network of several inflammatory cells and mediators. These include
antigen-presenting cells, T cells (mainly, Th2 type), mast cells,
basophils, eosinophils, and fibroblasts. Eosinophils represent the
major effector cells in allergic eye mechanism. Mast cells are normally
found in the substantia propria of the normal
conjunctiva.
1 Whereas mast cells are increased and
activated in the allergic conjunctiva and largely contribute to the
early-phase response of the allergic reaction, eosinophils are the main
cell type recruited during the late phase and represent the main factor
responsible for persistent inflammation and tissue damage, through
their preformed and newly synthesized mediators.
2 3 Once
in the tissues, eosinophils adhere, survive for several days, and
release their mediators, which cause tissue damage mainly detected when
the allergic inflammatory response becomes chronic. The cytokines
responsible for the prolonged eosinophil survival are interleukin
(IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor
(GM-CSF), also termed eosinophil survival cytokines.
4 5 6 7 8 9 10 Moreover, it has been recently shown that eosinophils can contribute to
the Th2-type cytokine profile of the allergic reaction through an
active production of IL-4, IL-5, and GM-CSF.
4
Fibroblasts are also involved in allergic eye diseases, particularly in
those manifesting persistent eosinophilic inflammation and giant
papillae, such as vernal keratoconjunctivitis.
3 Moreover,
fibroblasts are affected by inflammatory cells, and eosinophils have
been shown to induce lung and skin fibroblast proliferation and to
modulate their collagen production.
11 12 They represent
not only target cells responding to inflammatory stimuli with tissue
remodeling, but they may also contribute to modulation of allergic
inflammation in view of their ability to respond to and produce various
cytokines.
13 14 15 In particular, fibroblasts have been
shown to influence mast cell differentiation, survival, and functional
activity,
16 mainly by their ability to synthesize Stem
Cell Factor
17 and extracellular matrix components such as
fibronectin and laminin.
18 19 Studies have shown that
fibroblasts are also able to enhance eosinophil survival in vitro. In
fact, mouse 3T3 fibroblasts prolong human peripheral blood eosinophil
survival in the presence of exogenous GM-CSF.
6 Other
studies have shown that human lung fibroblasts enhance eosinophil
survival by their production of GM-CSF.
8 10
Relationships between eosinophils and fibroblasts are of great
importance for understanding the mechanisms of persistence of allergic
inflammation and tissue repair. Because eosinophils are found in
increased numbers in various allergic eye diseases in the conjunctiva,
in proximity to conjunctival fibroblasts, the interactions between
these two cell types could resemble those that have been demonstrated
to exist in lung and skin models.
The purpose of this study was to investigate the effect on eosinophil
survival and functional activity of coculturing human peripheral blood
eosinophils with human conjunctival fibroblasts. This coculture system
could serve as an in vitro model to investigate the interactions taking
place between eosinophils and fibroblasts in ocular allergic diseases.
Methods
Fibroblast Isolation and Culture from Conjunctival Biopsy Specimens
This research followed the tenets of the Declaration of Helsinki.
Conjunctival specimens were obtained by biopsy from the superior bulbar
conjunctiva in four patients during cataract surgery, after they
provided informed consent. None of these patients was treated with any
topical drug during the month preceding the surgery. The specimens were
cultured as explants in Dulbecco’s modified Eagle’s medium (DMEM,
Biological Industries, Beith Haemek, Israel) supplemented with 100 U/ml
penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, 1
mM HEPES, and 10% fetal calf serum (FCS; Biological Industries) and
incubated at 37°C in 5% CO2. The outgrown
fibroblasts were subcultured by treatment with 0.1% trypsin-0.02%
EDTA (Sigma, St. Louis, MO) for 5 minutes. For coculturing with
eosinophils, third-generation passaged fibroblasts were seeded in
96-well plates (Costar, Cambridge, MA) at 1 ×
104 cells per well in 300 μl DMEM-10% FCS.
Isolation and Purification of Eosinophils
Eosinophils were purified from four volunteers with mild atopia
(16–48 years of age) who were not at the time taking any oral
treatment for their condition and whose peripheral blood eosinophil
counts ranged from 4% to 10%. Informed consent was obtained from the
donors according to the guidelines established by the intramural
Hadassah–Hebrew University Human Experimentation Committee.
Eosinophils were purified from the peripheral blood as previously
described.
20 Briefly, venous blood (100–150 ml) was
collected in heparinized syringes. Blood was subjected to dextran 6%
(Sigma) sedimentation, and leukocytes were centrifuged on
Ficoll-Paque (density, 1.077; Sigma) for 25 minutes at
700
g at 4°C. Neutrophils in the granulocyte-enriched
pellet were tagged with micromagnetic beads to anti-CD16 antibodies,
lymphocytes were tagged with anti-CD3 antibodies, and mononuclear cells
with anti-CD14 antibodies (Miltenyi; Biotech, Bergisch Gladbach,
Germany). The tagged cells were then eliminated by passing them through
a magnetic field (MACS). Eosinophils were collected at a purity of 97%
to 100%, assessed by Kimura staining,
21 and at a
viability of more than 99%, assessed by trypan blue (Sigma) staining .
Coculture of Eosinophils with Conjunctival Fibroblasts
The medium of the fibroblast monolayers was gently aspirated, and
eosinophils (1 × 105 cells in 200 μl
enriched medium (EM) consisting of RPMI-1640 [Biological Industries]
supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, 1 mM HEPES, and 5% FCS) were added to each
well of confluent monolayers of conjunctival fibroblasts (96-well
plates) and incubated at 37°C in 5% CO2 for up
to 16 days. For extended periods of culturing, the culture medium was
changed every 4 days by gentle aspiration and addition of fresh medium.
Control samples consisted of eosinophils cultured in wells in the
absence of fibroblasts. For all the experiments, the cocultures were
repeated four times, each consisting of eosinophils and conjunctival
fibroblasts taken from four different individuals.
To assess whether the increased survival of eosinophils cultured with
fibroblasts was attributable to IL-3, IL-5, or GM-CSF, neutralizing
mouse anti-human monoclonal antibodies to these cytokines (R&D Systems,
Minneapolis, MN) were added to the fibroblast cultures when the
eosinophils were added (day 0 of the experiments). Antibodies were
added at a final concentration of 4 μg/ml each, found to be an
optimal concentration from pilot experiments in which two antibody
concentrations of 4 μg/ml and 40 μg/ml provided similar inhibition
values. In addition, to validate the neutralizing capacity of the
anti-cytokine antibodies, eosinophils were incubated with an optimal
concentration of recombinant human (rh)GM-CSF, rhIL-3, and rhIL-5 in
the presence of optimal neutralizing concentrations of, respectively,
anti-GM-CSF, anti-IL-3, and anti-IL-5. A nonrelevant monoclonal
antibody, human anti-CD3 antibody (Miltenyi) at a final concentration
of 4 μg/ml, was added as a control in these experiments.
In some experiments, freshly isolated eosinophils were cultured with
conjunctival fibroblast–conditioned medium. The conditioned medium was
obtained by culturing confluent monolayers of conjunctival fibroblasts
in DMEM-10% FCS for a period of 48 hours. To the eosinophils in
96-well plates (1 × 105/200 μl
EM) 200 μl conditioned medium was added, and the cells were
cultured for 96 hours.
Assessment of Eosinophil Viability
To assess the viability of eosinophils under the various culture
conditions, the trypan blue exclusion method was used as follows. To
evaluate viable eosinophils in coculture with conjunctival fibroblasts,
eosinophils were detached from the conjunctival fibroblast monolayer by
treatment with trypsin for 3 minutes. In this way, fibroblasts remained
adherent to the wells, while eosinophils detached from the fibroblast
monolayer. Twenty microliters of the detached eosinophils was withdrawn
from the wells by gentle resuspension. Trypan blue (20 μl) was added,
and the cells were immediately inspected in a hemocytometer under a
light microscope. Samples were examined blindly by two different
observers. The percentage of eosinophil viability was calculated as
follows: % viability of eosinophils = (number of cells excluding
the trypan blue as enumerated on the day of the test) · 100/(number
of cells seeded on day zero).
For eosinophils cultured in the absence of fibroblasts, 20 μl of the
cells were withdrawn after gentle resuspension, and examination of cell
viability was performed as described.
Assessment of eosinophil viability was performed after 4, 8, and 16
days of culture. The viability of eosinophils cultured in either
conditioned medium or of eosinophils cocultured with fibroblasts in the
presence of neutralizing antibodies was evaluated after 4 days of
culture.
Assessment of Eosinophil Adherence
The adherence of the eosinophils to the conjunctival fibroblast
monolayers was assessed after 4 days of coculture. To assess adherence,
wells were gently washed three times by the addition and aspiration of
300 μl EM. The remaining adherent eosinophils were counted under an
inverted microscope, using a grided eyepiece, at ×400 magnification in
at least 14 different fields. The number of adherent eosinophils was
calculated by multiplying the average number of cells per grid (counted
along a diameter of the well) by the number of times the well area
could be divided into the grid area. Data are given as percentage
adherent eosinophils, which is calculated as follows: % adherence = n/105 × 100, where n is
the average counted cells per grid × area of well/area of the
grid.
In some experiments, the role of either GM-CSF, IL-3, or IL-5 in
eosinophil adherence to fibroblasts was evaluated. Neutralizing
antibodies to any one of these cytokines (4 μg/ml) were added to the
eosinophil–fibroblast coculture on day 0, and adherent eosinophils
were evaluated 4 days later. Cocultures were inspected under an
inverted microscope on day 4, by three different observers, to account
for individual variability. These experiments were performed in
triplicate.
Eosinophil Peroxidase Determination after Eosinophil Activation
with Lipopolysaccharide
To assess their functional activity, eosinophils cocultured with
conjunctival fibroblasts were incubated on day 4 for 20 minutes at
37°C with 1 μg/ml lipopolysaccharide (LPS; Sigma). Fibroblast
monolayers in medium alone were also incubated with LPS (1 μg/ml), as
well as freshly isolated eosinophils seeded in wells in the absence of
fibroblasts immediately after purification (1 ×
10
5 cells/200 μl EM containing 1 μg/ml LPS).
After incubation, supernatants were collected, centrifuged (5 minutes,
120
g) and stored at −80°C until assessed. Eosinophil
peroxidase (EPO) release was determined by a colorimetric assay, as
previously described.
22 The substrate solution consisted
of 0.1 mM
O-phenylenediamine dihydrochloride (Sigma) in 0.05
M Tris buffer (pH 8.0) containing 0.1% Triton X-100 (Sigma) and 1 mM
hydrogen peroxide (Merck, Darmstadt, Germany). Aliquots (50 μl) of
the different supernatants were incubated with 50 μl substrate
solution for 10 minutes at room temperature. The reaction was stopped
by the addition of 100 μl of 4 mM sulfuric acid (BDH, Dorset, UK),
and the absorbance was determined at 490 nm by spectrophotometer.
Statistical Analysis
Results are expressed as mean ± SEM. Statistical analysis
was performed using the unpaired Student’s t-test or the
Mann–Whitney test where appropriate. P < 0.05 was
considered significant.
Results
Effect of Conjunctival Fibroblasts on Eosinophil Viability
As can be seen in
Figure 1 , survival of eosinophils cultured in medium alone was 5.8% ± 2.8%
and 0.52% ± 0.29% on days 4 and 8, respectively. Under these
conditions, no viable eosinophils were detected on days 12 and 16.
Addition of 50% fibroblast-conditioned medium to eosinophils did not
affect their survival. In fact, on the fourth day, 3.56% ± 0.59%
eosinophils were still alive in the conditioned medium. The viability
of eosinophils cultured on fibroblast monolayers was increased to
29.9% ± 1.9% on day 4 (
P = 0.0043, Mann–Whitney
test), to 12.8% ± 2.9% on day 8 (
P = 0.003), and to
3.4% ± 0.6% on day 12. On day 16, occasional eosinophils were still
surviving (0.7% ± 0.04%;
Fig. 1 ).
Effect of anti-6M-CSF, anti-IL-3 and anti-IL-5 Neutralizing
Antibodies on Eosinophil Viability and Adherence in Coculture
The increased survival of eosinophils in coculture with
conjunctival fibroblasts was partially inhibited by the addition of
either anti-GM-CSF, anti-IL-3, or anti-IL-5 antibodies
(Fig. 2) . In fact, eosinophil viability on day 4 decreased from 29.9% ± 1.9%
in coculture, to 11.8% ± 0.9% after addition of optimal
concentrations of anti-GM-CSF antibodies (
P = 0.019),
to 20.4% ± 1.4% with anti-IL-3 antibodies (
P =
0.033), and to 17.4% ± 0.7% with anti-IL-5 antibodies
(
P = 0.011). The nonrelevant antibody anti-CD3 did not
cause a significant change in eosinophil viability, compared with the
eosinophils cultured in medium alone (25.5% ± 3.8%,
P = 0.272).
Eosinophils were found by morphologic criteria to adhere quickly to the
conjunctival fibroblasts. In fact, they assumed a spindle shape instead
of presenting their round refractile appearance (not shown). On day 4,
75.3% ± 4.8% of the eosinophils in coculture were found to be
attached to the fibroblast monolayers, after extensive washing of the
cultures. Incubation of the coculture with an optimal concentration of
anti-GM-CSF antibodies decreased the adherence of the eosinophils to
the conjunctival fibroblasts to 57.9% ± 4.2% (P =
0.034). No significant changes in adherence were demonstrated when
either anti-IL3 or anti-IL5 was added to the cocultures. The addition
of anti-CD3 antibody to the coculture also did not affect eosinophil
adherence (85% ± 7.4%, P = 0.286).
Eosinophil Functional Activity in Coculture with Conjunctival
Fibroblasts
Next, we assessed whether eosinophils cocultured for 4 days with
conjunctival fibroblasts were functionally active. Cocultures were
incubated with the potent eosinophil stimulator, LPS, and the release
of EPO was evaluated as a measure of eosinophil activation.
Freshly isolated eosinophils incubated with an optimal concentration of
LPS (1 μg/ml) released EPO at levels of 0.51 ± 0.05 optic
density (OD; at 490 nm;
Table 1 ). Similarly, after 4 days of coculture with conjunctival
fibroblasts, eosinophils released EPO at 0.47 ± 0.04 OD,
indicating that the surviving eosinophils were functionally active.
Fibroblasts cultured for 4 days in medium alone, after 20 minutes’
incubation with LPS (1 μg/ml), expressed peroxidase activity of
0.21 ± 0.06 OD The EPO levels from freshly isolated eosinophils
cultured in medium alone was 0.11 ± 0.024 OD
(Table 1) .
To compare the levels of EPO released from activated fresh eosinophils
cultured alone with that in activated eosinophils cocultured with
conjunctival fibroblasts, the background peroxidase levels were
subtracted in these two groups as follows
(Table 1) . Peroxidase levels
found in activated fibroblasts cultured in medium alone were subtracted
from the EPO levels found in the coculture in each experiment. The mean
net EPO levels of eosinophils in coculture was calculated to be
0.26 ± 0.03 OD. Next, EPO levels found in nonactivated
eosinophils, were subtracted from the EPO levels found in the activated
eosinophils, in each experiment, giving a net value of 0.39 ±
0.07 OD, Because at day 4 only 29.9% of the initial eosinophils seeded
on conjunctival fibroblasts were viable, the EPO levels for the
cocultured eosinophils was calculated per 10
5 cells. Thus,
EPO levels found in activated eosinophils cocultured for 4 days with
conjunctival fibroblasts (0.87 ± 0.09 OD per 10
5 cells) was significantly higher than those secreted from activated
freshly isolated eosinophils (0.39 ± 0.07 per 10
5 cells;
P = 0.01, paired
t-test).
Discussion
In the present study, we have demonstrated that coculturing human
peripheral blood eosinophils with human conjunctival fibroblasts
results in prolonged survival, adherence, and enhanced functional
activity of eosinophils. Extended eosinophil survival in tissues seems
to be the key feature of allergic inflammation. In our coculture, 30%
of the initially seeded eosinophils were viable at day 4, and 13%
survived after 8 days in culture, whereas in medium alone only 5% were
still alive at day 4. In addition, we investigated the role of the
three survival cytokines for eosinophils—namely, IL-3, IL-5, and
GM-CSF—with this system. The addition of an optimal concentration of
neutralizing antibodies to either one of these three cytokines,
significantly inhibited the increased survival of eosinophils cultured
with conjunctival fibroblasts, indicating a role for the three of them.
Previous studies have shown increased survival of eosinophils when they
are cultured with mouse 3T3 fibroblasts,
6 human lung
fibroblasts,
8 and myofibroblasts from bronchial
mucosa
10 in the presence of GM-CSF. In these studies, the
fibroblast-induced prolonged survival of eosinophils was attributable
to exogenous GM-CSF and to the production of this cytokine by the
fibroblasts. However, in our study, in addition to the role of GM-CSF,
we found that IL-3 and IL-5 also contribute to increased eosinophil
survival.
The effects of the three cytokines (IL-3, IL-5, and GM-CSF) on
eosinophil survival in vitro was previously demonstrated by adding
these cytokines to cultured eosinophils.
5 7 9 Specifically, the addition of GM-CSF to eosinophils cultured with 3T3
fibroblasts was reported by Owen et al.
6 They demonstrated
an increased survival of eosinophils with GM-CSF alone, which was
further increased when the combination of GM-CSF and 3T3 fibroblasts
was tested.
The source of these three cytokines in our coculture system could be
autocrine secretion of the eosinophils or expression by the
conjunctival fibroblasts. Although GM-CSF is shown to be secreted from
fibroblasts, the evidence for IL-3 secretion from fibroblasts is
anecdotal,
23 and no evidence currently exists for the
ability of fibroblasts to secrete IL-5. In a recent ex vivo study on
eosinophil survival in nasal polyps, IL-5 was identified as a major
survival factor for eosinophils, and its source was located in
lymphocytes, mast cells, and eosinophils.
24 Currently, it
is not known whether conjunctival fibroblasts are capable of producing
any of the three cytokines studied. Several studies have demonstrated
the presence of IL-3, IL-4, IL-5, IL-6, IL-13, and tumor necrosis
factor-α in biopsy specimens of conjunctiva from patients with
allergic eye disease.
25 26 27 However, the source of these
cytokines was related to mast cells, T lymphocytes, eosinophils, and
conjunctival epithelial cells. Therefore, we can assume that in our
system, GM-CSF could have been secreted from both fibroblasts and
eosinophils, whereas the secretion of IL-5 and IL-3 could be an
autocrine contribution by the eosinophils alone, stimulated by their
contact with the fibroblast monolayers.
It is important to point out that other factors may be involved in the
survival of eosinophils cocultured with conjunctival fibroblasts. In
fact, an optimal concentration of anti-IL-3, anti-IL-5, and anti-GM-CSF
neutralizing antibodies did not completely inhibit the survival of
eosinophils. It is therefore conceivable that other mediators, produced
by the fibroblasts and the eosinophils, such as tumor necrosis
factor-α,
28 stem cell factor,
29 fibronectin, and laminin,
30 31 could also contribute to
this effect.
Interestingly, we could not demonstrate an increased viability of
eosinophils when cultured with fibroblast-conditioned medium. The
absence of effect of the conditioned medium is in agreement with
previous studies
6 10 but in contrast with
another.
8 The absence of effect of the conditioned medium
implies that adhesion is necessary for both eosinophils and fibroblasts
to secrete one or more of the three survival cytokines. Adhesion of
eosinophils to tissue fibronectin has been shown to result in a
significant autocrine production of GM-CSF, but not of
IL-5,
30 and blocking of β-1 integrin adhesion molecules
on eosinophils reduced their survival in coculture.
10
In addition to the increased survival of eosinophils, coculture with
conjunctival fibroblasts enhanced their functional activity after 4
days in culture, when compared with that of freshly isolated
eosinophils. In fact, it was found that EPO levels secreted after LPS
activation of the cocultured eosinophils were significantly higher than
after activation of freshly isolated eosinophils. This increased
activity can again be explained by the contact with conjunctival
fibroblasts. Indeed, eosinophil adhesion was recently demonstrated to
be a crucial step in the activation, signaling, and functional activity
of these cells.
32 In addition, GM-CSF can also take part
in priming eosinophils to release preformed mediators.
9 6 A recent study demonstrated a crucial role for GM-CSF secreted by human
lung fibroblasts in increasing the activity of eosinophils as
manifested by increased CD11b and decreased L-selectin expression. A
combined mechanism of cell contact and cytokine release was
suggested.
33
In our coculture, eosinophils adhered to the fibroblast monolayers.
This adherence was reduced by addition of anti-GM-CSF but was unchanged
after the addition of either anti-IL-3 or anti-IL-5 to the system. It
has been previously shown that both GM-CSF and IL-5 induce adhesion
molecule expression on eosinophils and their adherence to endothelial
cells.
28 However, a number of studies point out a central
role for GM-CSF in enhancing adherence of eosinophils to tissue
components. We have recently found that eosinophils incubated for 3
days in medium containing 10 ng/ml GM-CSF, demonstrated increased
expression of VLA-4 (92.5% of the cells were positive for VLA-4,
compared with 9.4% when the cells were incubated in medium alone;
Temkin V, Hartman M-L, Levi-Schaffer F, unpublished data, February
1999). In another study, incubation of eosinophils with GM-CSF,
resulted in increased expression of the adherence molecule CD11b by
eosinophils, and was associated with increased adherence of the cells
to gelatin-coated plastic.
34 In addition, GM-CSF, but not
IL-5 and IL-3, is the main cytokine involved in the enhanced survival
shown by eosinophils adherent to laminin-coated wells.
31 A
possible explanation for the failure of anti-IL-5 to inhibit adherence
of eosinophils to conjunctival fibroblasts may be the absence of
secretion of this cytokine from conjunctival fibroblasts in our system.
In conclusion, we have shown that coculture of human peripheral blood
eosinophils with conjunctival fibroblasts influences eosinophil
survival, adherence, and activation, and that GM-CSF is a key modulator
of these effects. The increased viability and functional activity of
eosinophils cocultured with conjunctival fibroblasts is probably a
result of cellular contact and adhesion combined with cytokines
released from both cell types as a result of this contact. This
coculture can serve as an in vitro model for the study of
eosinophil–fibroblast interactions in allergic eye diseases,
especially vernal keratoconjunctivitis, in which a persistent
eosinophilic inflammation is combined with intense fibroblast
proliferation. In addition, the methodology used in this work may serve
as a useful model for a better understanding of the relationship
between eosinophils and fibroblasts in other allergic diseases, such as
asthma, characterized by eosinophil infiltration and tissue remodeling.
Supported by a grant from The Joint Research Fund of The Hebrew University and Hadassah University Hospital, Jerusalem, Israel.
Submitted for publication July 8, 1999; revised October 26, 1999; accepted November 8, 1999.
Commercial relationships policy: N.
Corresponding author: Francesca Levi–Schaffer, Department of Pharmacology, Hadassah Medical School, The Hebrew University, PO Box 12065, Jerusalem 91120, Israel.
fls@cc.huji.ac.il
Table 1. EPO Release from Eosinophils After LPS Activation
Table 1. EPO Release from Eosinophils After LPS Activation
| Culture Type | Culture Time | Total Peroxidase Levels Measured | Net Levels | Corrected Levels per 105 Cells |
| E in medium | Freshly isolated eosinophils | 0.11 ± 0.02 | — | — |
| E+ LPS | Freshly isolated eosinophils | 0.51 ± 0.05 | 0.39 ± 0.07* | 0.39 ± 0.07 |
| F+ LPS | 4 days | 0.21 ± 0.06 | — | — |
| E+ F+ LPS | 4 days | 0.47 ± 0.04 | 0.26 ± 0.03, † | 0.87 ± 0.09 (P = 0.01) |
The authors thank Dalia Pickholz for her expert technical
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