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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 694-702
The Biologic Role of Interleukin-8: Functional Analysis and
Expression of CXCR1 and CXCR2 on Human Eosinophils
By
Holger Petering,
Otto Götze,
Daniela Kimmig,
Regina Smolarski,
Alexander Kapp, and
Jörn Elsner
From the Department of Dermatology and Allergology, Hannover Medical
University, Hannover, Germany; and the Department of Immunology,
Georg-August University of Göttingen, Göttingen, Germany.
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ABSTRACT |
Chemokines play an important role in attracting granulocytes into
sites of inflammation. Two chemokine subfamilies differ in their
biologic activity for different granulocyte subsets. Whereas CXC
chemokines such as interleukin-8 (IL-8) activate predominantly neutrophils, CC chemokines such as RANTES and eotaxin activate predominantly eosinophils. However, controversial results have been
published in the past regarding the biologic role of IL-8 in eosinophil
activation, particularly in allergic diseases. In this study, we
investigated the functional evidence and expression of both IL-8
receptors, CXCR1 and CXCR2, on highly purified human eosinophils. In
the first set of experiments, a chemotaxis assay was performed showing
that IL-8 did not induce chemotaxis of eosinophils. In addition, and in
contrast to neutrophils and lymphocytes, IL-8 did not induce a rapid
and transient release of cytosolic free Ca2+
([Ca2+]i) in eosinophils, even after
preincubation with TH1- and TH2-like cytokines. To investigate whether
neutrophil contamination might be responsible for the reported IL-8
effects on eosinophils, neutrophils were added to highly purified
eosinophils from the same donor in different concentrations.
Interestingly, as little as 5% of neutrophil contamination was
sufficient to induce an increase of [Ca2+]i
after stimulation with IL-8. Flow cytometry experiments with monoclonal
antibodies against both IL-8 receptors demonstrated no expression of
CXCR1 and CXCR2 on eosinophils before or after cytokine activation.
Reverse transcriptase-polymerase chain reaction experiments showed that eosinophils, in contrast to neutrophils and
lymphocytes, did not express mRNA for CXCR1 and CXCR2. In summary, this
study clearly demonstrates that CXCR1 and CXCR2 are not expressed on
human eosinophils, even after priming with different bioactive
cytokines. Because the CXC chemokine IL-8 did not induce in vitro
effects on human eosinophils, IL-8 may also not contribute in vivo to
the influx of eosinophil granulocytes into sites of allergic
inflammation. Our results suggest that CC chemokines such as eotaxin,
eotaxin-2, and MCP-4 are predominant for the activation of eosinophils.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CHEMOKINES PLAY an important role in
attracting granulocytes into sites of inflammation. Up to now, four
different subfamilies of chemokines have been identified according to
highly conserved cysteine motifs in their aminoterminal
domain.1,2 The two major subfamilies of chemokines, CXC and
CC chemokines, differ in their biologic activity to stimulate different
kinds of effector cells. Whereas CXC chemokines such as interleukin-8 (IL-8), NAP-2, GRO- , - , - , and ENA-78 activate predominantly neutrophils,3 CC chemokines such as RANTES, MCP-4, and
eotaxin activate eosinophils, basophils, and, as described recently,
T-lymphocyte subsets.4-6 However, controversial results
have been published in the past regarding the biologic importance of
IL-8 in eosinophil activation, particularly in allergic diseases such
as bronchial asthma and atopic dermatitis but also in
hypereosinophilia, eg, in Hodgkin's disease.
Competition-binding studies showed that human neutrophil granulocytes
bear two classes of IL-8 receptors, CXCR1 (IL-8RA)7 and
CXCR2 (IL-8RB).8,9 Both receptors are binding IL-8 with high affinity in contrast to the other CXC chemokines, NAP-2 and GRO-, which bind with high affinity only to CXCR2.10
Thus, CXCR1 is referred to as an IL-8-specific receptor, whereas CXCR2 is regarded as a promiscuous receptor responding to various CXC chemokines. Jones et al11 demonstrated that CXCR1 and CXCR2 are functionally different due to aminoacid sequence differences clustered at the NH2- and COOH-terminal domains. Transient
changes of cytosolic free Ca2+ and the release of granular
enzymes were mediated by both receptor types, whereas the production of
reactive oxygen species via the NADPH oxydase depended exclusively on
stimulation through CXCR1.11 Wuyts et al12
characterized granulocyte chemotactic protein 2 (GCP-2) as another CXC
chemokine signaling through CXCR1 and CXCR2 and being nearly as
effective as IL-8. As opposed to human neutrophils, they found no
evidence for any activity on human eosinophils.12
Donnelly et al13 reported that, in patients with an early
stage of adult respiratory distress syndrome, serum concentrations of
IL-8 could be detected in picomolar ranges and, therefore, initiate the
migration of granulocytes towards the inflamed area. In the sputum of
patients with chronic inflammatory airways disease, typically
associated with serum and tissue eosinophilia, concentrations of IL-8
were reported to range from 1 to 9 nmol/L. But these studies did not
verify a direct effect of IL-8 on human eosinophils. The importance of
the CXC chemokine IL-8 for eosinophil activation and the expression of
CXCR1 and CXCR2 on human eosinophils are, therefore, still a matter of
debate.
After priming with the eosinophil-specific cytokine IL-5, Schweizer et
al14 reported that stimulation of human eosinophils with
IL-8 did induce chemotaxis and actin polymerization as related events.
Their cell preparations consisted of up to 95%
eosinophils.14 In another study, IL-8 was found to be a
chemoattractant for eosinophils purified from patients with blood
eosinophilia. It was proposed that this might be due to in vivo priming
mechanisms.15 These data are in contrast to previous
reports in which the effect of IL-8 on human eosinophils was found to
be negligible.16,17
Eosinophils are known to produce and secrete IL-8,18-21
which can be stimulated by TH2 cell-derived cytokines.22
Therefore, it may be assumed that the enhanced production of bioactive
IL-8 after priming the cells with cytokines results in a downregulation of CXCR1 and CXCR2 on eosinophils.23 Schnyder-Candrian et
al24 found that interferon (INF) inhibits the
production of IL-8 and ENA-78 in human monocytes.
In this study, we investigated the functional evidence and expression
of both IL-8 receptor types, CXCR1 and CXCR2, on human eosinophils from
healthy nonatopic volunteers. In addition, purified eosinophils were
primed with TH1 and TH2 cell-derived cytokines. To investigate whether
neutrophil contamination might be responsible for the reported IL-8 in
vitro effects on eosinophils, neutrophils were added to highly purified
human eosinophils from the same donor in various concentrations and
functional assays were performed. Therefore, this study helps to
understand the effects of IL-8 on human eosinophils and may help to get
insight into the physiologic role of this chemokine during the
inflammatory process.
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MATERIALS AND METHODS |
Isolation of human eosinophils.
Human granulocytes were isolated from heparin-anticoagulated venous
blood from normal nonatopic healthy European donors without signs of
bacterial or viral infections. All donors were nonsmokers and did not
take any medicine. The isolation was performed using Ficoll (Pharmacia,
Uppsala, Sweden) density gradient centrifugation as
described previously.25 For further purification,
granulocytes were resuspended in HEPES-buffered Hanks' Balanced Salt
Solution (HBSS; GIBCO, Grand Island, NY), pH 7.4, containing 1 mg/mL
bovine serum albumin (BSA; HBSS + BSA). Eosinophils were purified by negative selection with anti-CD16 antibody (clone 3G8; Immunotech, Hamburg, Germany) coated Dynabeads M-450 (Dynal, Hamburg, Germany), as
described previously.25 The resulting eosinophil purity was  99.5% as determined by flow cytometrical analysis (FACScan; Becton Dickinson, Heidelberg, Germany) using phycoerythrin-conjugated anti-CD16 antibody (clone 3G8; Immunotech).
Priming of eosinophils with different cytokines.
For some experiments, highly purified human eosinophils were incubated
for 24 or 36 hours at 37°C with 50 ng/mL IL-4, 50 ng/mL IL-5, 30 ng/mL tumor necrosis factor (TNF), 100 ng/mL INF, 100 ng/mL
granulocyte-macrophage colony-stimulating factor (GM-CSF), or medium
alone; washed; and resuspended in assay buffer before the assessment of
their functional response to IL-8. Viability after incubation was
determined by Kimura staining and always 85%.
Monoclonal antibodies (MoAbs).
The human anti-CXCR1 MoAb (MoAb SE2) and human anti-CXCR2 MoAb (MoAb
HC2) were used as described previously.26 The human IgG1
and IgG1-fluorescein isothiocyanate (FITC)-conjugated
isotype control; human anti-CD3, anti-CD4, anti-CD8, and anti-CD19
MoAbs; and also the human IgG2b and IgG2b-FITC-conjugated isotype
control were obtained from Sigma Chemicals (Deisenhofen, Germany). The humanized anti-CD52 MoAb (Campath-1H monomer) was a kind gift from
Wellcome Foundation (London, UK).27
Immunofluorescence of granulocytes and eosinophils.
Immunofluorescence of granulocytes was performed with standard
techniques. In brief, granulocytes and eosinophils were adjusted to a
density of 1 × 107 cells/mL. Aliquots (20 µL)
containing 2 × 105 cells were incubated at 4°C
for 30 minutes with the indicated antibody. Thereafter, cells were
washed twice with cold phosphate-buffered saline. For indirect
immunofluorescence, cells were stained in a second step with an
FITC-conjugated mouse antihuman antibody (Immunotech) and subsequently
washed twice. In some experiments, double staining with FITC-conjugated
antibodies and anti-CD16 phycoerythrin (PE)-conjugated antibody or
anti-CD3, CD4, CD8, and CD19 PE-conjugated antibodies were performed.
Thereafter, cells were analyzed by flow cytometry (FACScan). The sample
was excited at 488 nm and emission was measured at 530 nm (FITC-labeled antibodies, Fluorescence 1, green fluorescence) and at 585 nm (anti-CD16 PE, Fluorescence 2, red fluorescence).
Eosinophils were preincubated for 24 and 36 hours in the presence of
100 ng/mL INF, 50 ng/mL IL-4, 50 ng/mL IL-5, 30 ng/mL TNF, and
100 ng/mL GM-CSF (Genzyme, Rüsselshiver, Germany)
and RPMI medium, respectively. Thereafter, cells were washed and
stained by anti-CXCR1 MoAb, CXCR2 MoAb, or isotype control.
CXCR1 and CXCR2 mRNA expression.
Total RNA was isolated from eosinophils, lymphocytes, and neutrophils
using TRIzol (GIBCO) according to the manufacturer's instructions
based on the guanidine isothiocyanate method. First-strand cDNA
synthesis was performed in a 20 µL reaction mixture containing 5 µL
RNA, 1 mmol/L dNTP, 1.6 µg Oligo-p(dT)15 primer, 50 U
RNase inhibitor, 20 U avian myeloblastosis virus (AMV)
reverse transcriptase (Boehringer Mannheim, Mannheim, Germany),
incubated at 25°C for 10 minutes, and then incubated at 42°C
for 1 hour. The AMV reverse transcriptase was denatured by 99°C for
5 minutes and then placed on ice. Primers for the amplification of
CXCR1 (sense, 5 -CGACTGTGGGCGGATTCTTG-3; and antisense,
5-AGACCGATACCATGTGCTCT-3), CXCR2 (sense,
5-ACGCATGTTGCTGTCTCTGG-3; and antisense,
5-TGTTGGTCCTAGGGCGTAG-3), CD16 (sense,
5-GGCCTCGAGCTACTTCATTG-3; and antisense,
5-GGAGCCGCTATCTTTGAGTG-3), and CD52 (sense,
5-GCCACGAAGATCCTACCAAA-3; and antisense,
5-GCTTGGCCCCTACATCATTA-3) were designed according to the
published sequences (accession numbers are as follows: CXCR1, L19591;
CXCR2, M73969; CD16, M24854; CD52, A23013). Reverse transcriptase
reaction mixture was used in the polymerase chain reaction (PCR) in a
50 µL final volume, 0.2 mmol/L of each dNTP, 0.4 µmol/L of each
primer (CXCR1, CXCR2, CD16, or CD52), and 1.3 U Taq DNA polymerase
(Boehringer Mannheim). The mixture was incubated in a thermocycler
using the following temperature profile: initial denaturation step at
94°C for 5 minutes, followed by 35 cycles of denaturation at
94°C for 30 seconds, annealing at 58°C for 30 seconds, and
extension at 72° C for 1 minute. The final extension step was at
72°C for 5 minutes. PCR samples were run on a 1.8% agarose gel
stained with 0.2 µg/mL ethidium bromide, and the PCR products were
visualized with UV light and photographed.
Chemotaxis assay.
In analogy to the previously described modified Boyden chamber
technique,16,28-30 the chemotaxis of human eosinophils,
neutrophils, and lymphocytes was determined by filling the lower
chamber with the stimuli and the upper chamber with the cells. A
polycarbonate membrane of 3 µm pore size was used for eosinophils and
neutrophils. For lymphocytes, the polycarbonate membrane had a pore
size of 1 µm. Human eosinophil, neutrophil, or lymphocyte suspensions of 100 µL at a concentration of 5 × 105/mL cells
were placed in the upper part of each chamber and migration was allowed
to proceed for 1 hour in a humidified atmosphere at 37°C. The lower
part of the Boyden chambers contained the migrated cells that were
subsequently lysed by adding 0.1% Triton X-100. Using
p-nitrophenyl -D-glucuronide (Sigma Chemicals) as a
substrate, the -glucuronidase activity in the lysates was measured
photometrically. Fluorescence readings were taken on a Titertek
Twinreader Plus (EFLAB, Finland) with 405 nm emission wave
length. For calculation of the number of migrated cells based on the
-glucuronidase activity determined in the lower part of the Boyden
chamber, values were calculated by a computer-assisted technique from a
standard curve using known numbers of unchallenged eosinophils. The
assays were performed in duplicates or triplicates. The results were
expressed as a ratio between the number of migration cells in the
sample versus the control medium, which reflects spontaneous migration. This ratio is referred to as chemotactic index (CI).
Measurement of [Ca2+]i in
spectrofluorometry.
For the measurement of the cytosolic free Ca2+
concentration ([Ca2+]i) of human eosinophils,
neutrophils, and lymphocytes, the fluorescence Ca2+
indicator Fura-2 (Molecular Probes, Eugene, OR) was used at a concentration of 2 µmol/L. Fluorescence was detected in an Aminco Bowman Series 2 spectrofluorometer (SLM-Aminco, Urbana, IL), as described previously.31,32 In brief, after addition of each stimulus and subsequent measurement, maximal and minimal fluorescence intensities were calibrated by the addition of 0.2% Triton X-100 leading to 100% Fura-2 saturation followed by a subsequent quenching of the fluorescence with 2 mmol/L EGTA. Fura-2 fluorescence changes were continuously monitored at a dual excitation spectra at
 1 = 340 nm and 2 = 380 nm; the emission
wavelength was fixed at 510 nm.32 Absolute
[Ca2+]i was calculated automatically by AB 2 series 2 software (SLM-Aminco) according to the equation described by
Grynkiewicz et al.33
Statistical analysis.
Unless otherwise stated, the data in the text and figures are expressed
as the mean ± SEM analysis of variance (ANOVA). Newman-Keuls tests
were used for comparing experimental groups to control values. P values less than .05 were accepted as significant. When the global test of differences was significant at the 5% level, pairwise tests of differences between groups were applied (Student's
t-test for paired data using 5% significance level, closed
test procedure).
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RESULTS |
IL-8 does not induce chemotaxis of human eosinophils, whereas human
neutrophils and lymphocytes are stimulated.
To investigate whether IL-8 stimulates highly purified human
eosinophils, the modified Boyden chamber technique was performed to
detect the chemotactic activity of IL-8 in comparison to the potent
eosinophil activator eotaxin and C5a. As seen in
Fig 1, IL-8 did not induce a significant
response of eosinophils at concentrations known to be potent for
lymphocytes and neutrophils. Also, higher or lower concentrations of
IL-8 did not induce a chemotactic response in eosinophils. In contrast
to eosinophils, IL-8 induced significantly chemotaxis of human
neutrophils and lymphocytes (Fig 1). The CC chemokines eotaxin, RANTES,
and C5a were used as positive controls. They induced chemotaxis in the
different cell types in expected patterns. Therefore, the CXC-chemokine
IL-8 is not a chemotactic stimulus for human eosinophils.
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| Fig 1.
IL-8 induces chemotaxis of human neutrophils and
lymphocytes but does not stimulate eosinophils. The chemotactic
activity was measured using the modified Boyden chamber technique and
expressed as the CI, defined as the ratio of the number of migrating
cells in presence of stimulus versus migrating cells in the presence of
medium. Cells were incubated with the indicated stimuli. The results
are presented as the mean ± SEM of five different experiments. ( )
Eosinophils; ( ) neutrophils; ( ) lymphocytes.
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Effect of IL-8 on [Ca2+]i in human
eosinophils.
To further evaluate whether IL-8 induces eosinophil activation, changes
in [Ca2+]i were investigated using the
fluorescence Ca2+ indicator Fura-2. Stimulation of highly
purified human eosinophils with IL-8 did not induce an increase in
[Ca2+]i (Fig 2).
In addition, preincubation of highly purified eosinophils with 50 ng/mL
IL-4, 50 ng/mL IL-5, 30 ng/mL TNF, 100 ng/mL GM-CSF, or 100 ng/mL
INF for 24 or 36 hours, respectively, and subsequent stimulation
with IL-8 with various concentrations also did not induce
[Ca2+]i transients in eosinophils (data not
shown). In contrast to eosinophils, human neutrophils and lymphocytes
showed rapid and transient changes of [Ca2+]i
after stimulation with IL-8 (Fig 2).
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| Fig 2.
IL-8 induces [Ca2+]i
transients in human neutrophils and lymphocytes but not in eosinophils.
Spectrofluorometric measurements of [Ca2+]i
in Fura-2-loaded purified human eosinophils, neutrophils, and
lymphocytes were performed. Cells were stimulated at the indicated time
points; C5a or RANTES was used as a positive control. One
representative experiment of six performed is shown.
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The purity of eosinophil preparations is important for receptor
studies.
To rule out the influence of neutrophils contaminating the preparation
of eosinophil granulocytes on measurements of
[Ca2+]i, further experiments with
Fura-2-loaded cells were performed. Different amounts of human
neutrophils were added to a granulocyte suspension of highly purified
human eosinophils from the same donor and subsequently stimulated with
IL-8 and eotaxin. Changes in [Ca2+]i were
detected in cell suspensions containing 100% highly purified human
eosinophils down to 0%, containing only purified neutrophils. As seen
in Fig 3A, a detectable but low neutrophil
contamination of 5% in the granulocyte suspension resulted in
[Ca2+]i transients. Therefore, 50,000 contaminating neutrophils do induce detectable differences in
[Ca2+]i. In contrast to IL-8, eotaxin was
highly effective to induce [Ca2+]i transients
in this granulocyte suspension (Fig 3A). Increasing numbers of
contaminating neutrophils, up to 100% in the granulocyte suspension,
resulted in higher increase of [Ca2+]i in
response to IL-8 but not to eotaxin (Fig 3A and B).
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| Fig 3.
(A and B) Contaminating neutrophils within preparations
of human eosinophils are leading to [Ca2+]i
transients. Spectrofluorometric measurements of
[Ca2+]i in Fura-2-loaded unprimed human
eosinophils between 100% and 0% purity were performed. All
contaminating cells were neutrophils as detected by flow cytometry. (A)
Eosinophils contaminated with different percentages of neutrophils were
stimulated with 100 ng/mL IL-8 and subsequently received 500 ng/mL
eotaxin as a positive control. One representative experiment of five
performed is shown. (B) Statistical analysis of
[Ca2+]i in human eosinophils contaminated
with different amounts of purified human neutrophils (0% up to 100%).
Cells were stimulated with 100 ng/mL IL-8 or 100 ng/mL eotaxin as a
positive control. The results are presented as the mean ± SEM of five
experiments.
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CXCR1 and CXCR2 are not expressed on the surface of human
eosinophils.
In the next set of experiments, the expression of both IL-8 receptor
types, CXCR1 and CXCR2, on human eosinophils was investigated by flow
cytometry. Highly purified human neutrophils were used as a positive
control and stained with anti-CXCR1 MoAb (SE 2) and anti-CXCR2 MoAb (HC
2) in different concentrations (0.5 to 50 µg/mL). Histogram analysis
showed binding of both MoAbs to human neutrophils. Maximal binding of
anti-CXCR1/CXCR2 MoAbs was reached at 20 µg/mL
(Fig 4) and showed that CXCR1 and CXCR2 are expressed on human neutrophils. The second proportion of cells appearing negative for CXCR1 and CXCR2 were human eosinophils, as
detected by staining with anti-CD52 MoAb. In addition, highly purified
human lymphocytes could also be stained with CXCR1/CXCR2 MoAbs
indicating the expression of both IL-8 receptor types (Fig 4). Two cell
populations are seen with a proportion of lymphocyte subsets negative
for CXCR1 and CXCR2. Double-color flow cytometric analysis showed that
CXCR1 and CXCR2 are expressed on CD8+ T cells, but not on
CD19+ B lymphocytes and CD4+ T cells (data not
shown). These data are in accordance with previous findings.34-37 In contrast to human neutrophils and
lymphocytes, highly purified CD16 selected human
eosinophils did not express CXCR1 and CXCR2 (Fig 4). Furthermore,
priming of human eosinophils for 24 or 36 hours, respectively, with 50 ng/mL IL-4, 50 ng/mL IL-5, 30 ng/mL TNF, 100 ng/mL GM-CSF, or 100 ng/mL INF, respectively, did not induce the expression of CXCR1 or
CXCR2 on the surface of eosinophils (Table
1).
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| Fig 4.
CXCR1 and CXCR2 are expressed on human neutrophils and
lymphocytes but not on human eosinophils. Flow cytometric analysis of
human neutrophils, lymphocytes, and eosinophils. Cells were
double-stained with the anti-CXCR1 or anti-CXCR2 MoAbs, respectively,
and anti-CD3, CD4, CD8, and CD19 MoAbs for lymphocyte subsets;
anti-CD16 MoAb for neutrophils; or anti-CD52 MoAb for eosinophils,
respectively. One representative experiment of eight performed is
shown.
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Table 1.
Expression of CXCR1 and CXCR2 on Human Neutrophils,
Lymphocyte Subsets, and Eosinophils From Healthy Nonatopic Donors
Stimulated by the Indicated Cytokines
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CXCR1 and CXCR2 mRNA are not expressed in human eosinophils.
To further confirm the binding results of anti-IL-8 receptor MoAbs,
reverse transcriptase-PCR (RT-PCR) was performed to
investigate whether purified human eosinophils express mRNA specific
for the IL-8 receptors CXCR1 and CXCR2. Again, human neutrophils and
lymphocytes were used as positive controls. As seen in
Fig 5, no
CXCR1 or CXCR2 mRNA could be detected in highly purified human
eosinophils. In addition, preincubation of human eosinophils for 24 or
36 hours with 100 ng/mL INF, 50 ng/mL IL-4, 50 ng/mL IL-5, 30 ng/mL
TNF, or 100ng/mL GM-CSF, respectively, did not induce the expression of CXCR1/CXCR2 mRNA (data not shown). Therefore, mRNA specific for
CXCR1 or CXCR2 is not expressed by human eosinophils. As expected and
in contrast to eosinophils, highly purified human neutrophils and
lymphocytes did express constitutively CXCR1 and CXCR2 mRNA, as
indicated by specific RT-PCR products with an expected size of 507 bp
for CXCR1 and 520 bp for CXCR2, respectively (Fig 5). To demonstrate
that all mRNA preparations were highly purified and not contaminated
with other cell types, RT-PCR was performed simultaneously using
primers for CD16 (neutrophils) and CD52 (eosinophils and lymphocytes)
(Fig 5).
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| Fig 5.
CXCR1 and CXCR2 mRNA is expressed in human neutrophils
and lymphocytes but not in human eosinophils. Human neutrophil,
lymphocyte, and eosinophil mRNA was isolated and first-strand cDNA
synthesis was performed. PCR was performed with primer pairs specific
for CXCR1 (expected size, 507 bp), CXCR2 (expected size, 520 bp), CD52
(expected size, 385 bp), and CD16 (expected size, 297 bp). The
amplicons were separated by electrophoresis in 1.8% agarose gel and
stained by ethidium bromide. Lane M, 100-bp DNA size marker; lane N,
human neutrophil mRNA; lane L, human lymphocyte mRNA; lane E, human
eosinophil mRNA. One representative experiment of four performed is
shown.
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| |
DISCUSSION |
The CXC chemokine IL-8 has been shown to play a central role in several
chronic inflammatory diseases, such as allergic bronchial asthma,38 rheumatoid arthritis,39,40 and
psoriasis.41 The recruitment and activation of human
neutrophil granulocytes is especially important for the inflammatory
response in these disorders. It could be demonstrated that IL-8 acts
via specific G-protein-coupled receptors, named CXCR1 and CXCR2, which
are expressed on the surface of human neutrophils, lymphocyte subsets,
and keratinocytes. In addition, IL-8 has been found in the BAL of
patients with allergic bronchial asthma and it has, therefore, been
speculated that IL-8 may also be responsible for the attraction of
eosinophils that are the predominant cells in BAL of asthmatic
patients. To clarify the controversial data concerning the effects of
IL-8 on human eosinophils, we investigated the in vitro effect of IL-8
and the expression of both IL-8 receptors on human eosinophils in
comparison to neutrophils and lymphocytes.
In the first set of experiments, the functional role of IL-8 on human
eosinophils was investigated. We found that IL-8 did not stimulate
chemotaxis and [Ca2+]i transients of highly
purified eosinophil granulocytes from healthy nonatopic donors. These
data indicate that IL-8 does not play a role in the recruitment of
eosinophils to the site of inflammation and are contrasting previous
results that IL-8 may be a potent chemotaxin for human eosinophils.
Erger and Casale42 have demonstrated that IL-8 is a potent
mediator of eosinophil chemotaxis through umbilical vein endothelial
cell and human pulmonary type II-like epithelial cell monolayers
cultured on polycarbonate filters. However, their eosinophil
preparations had only an average purity of 78%, with a maximal
chemotactic response of 25% of the granulocyte suspension through
naked filters. In addition, Sehmi et al15 reported that
eosinophils exhibit chemotaxis towards IL-8. Again, the investigators
in that study used cell preparations consisting up to only 80% ± 4% and 92% ± 2% eosinophils from normal donors and subjects with
hypereosinophilia, respectively. Further studies describing the
response of human eosinophils toward IL-8 also used eosinophil
preparations with only up to 95% purity.14,43-45
Therefore, it may be speculated that the in vitro effects in these
studies could be due to contaminating human neutrophils. To address
this issue, neutrophils were added to various preparations of highly
enriched human eosinophils from the same donor. We found that as little
as 5% neutrophil contamination was enough to transient changes in
[Ca2+]i. Thus, these data indicate that the
previously published effects of IL-8 on eosinophil chemotaxis may be
due to a contamination with neutrophils. They further point out that
highly purified eosinophils are essential for studying the in vitro
effects of cytokines and receptor expression in these cells.
To investigate whether in vivo priming mechanisms of human eosinophils
may be responsible for the expression of CXCR1 and CXCR2, highly
purified human eosinophils were preincubated with different cytokines
known to alter eosinophil activation: IL-4 and IL-5 were used as TH2
cell-derived cytokines well-known as specific activators of eosinophil
granulocytes46,47 and capable of stimulating eosinophils to
synthesize and release cytokines and express cytokine receptors on the
cell surface. Previously, Sehmi et al15 have reported that
eosinophils exhibit functional responses toward IL-8 in contrast to
cells from normal healthy donors due to in vivo priming mechanisms and
identified IL-5 as a cytokine enhancing the chemotactic response to
IL-8. In another study, IL-5-primed eosinophils showed a chemotactic
response to IL-8.14 However, our data showed that neither
IL-4, TNF, INF, and IL-5 nor GM-CSF was able to prime eosinophils
to induce [Ca2+]i transients in response to
IL-8.
In the last set of experiments, the expression of IL-8 receptors on the
surface of human eosinophils was investigated. Flow cytometric
measurements showed that eosinophils did not express CXCR1 or CXCR2 on
the cell surface. Moreover, priming of eosinophils with cytokines such
as IL-4, IL-5, TNF, IFN, and GM-CSF had no effect on the
expression of IL-8 receptors. Furthermore, RT-PCR analysis did not show
any mRNA transcripts specific for CXCR1 or CXCR2 in human eosinophils,
whereas in human neutrophils and lymphocytes, mRNA specific for both
IL-8 receptor types was detected.
In summary, this study demonstrates that CXCR1 and CXCR2 are not
expressed on human eosinophils even after priming with different bioactive cytokines. Although eosinophils produce IL-8 itself, the CXC
chemokine IL-8 seems to have no direct effect on eosinophils and there
is no evidence for an autocrine stimulation of eosinophils in the state
of hypereosinophilia. Because the CXC chemokine IL-8 did not induce
in vitro effects on human eosinophils, IL-8 may also not contribute in
vivo to the influx of eosinophils into sites of allergic inflammation.
Our results suggest that CC chemokines such as eotaxin, eotaxin-2, and
MCP-4 as well as their receptor CCR3 are predominant for the activation
of eosinophils.
| |
FOOTNOTES |
Submitted March 31, 1998;
accepted September 23, 1998.
Supported by a grant from the Deutsche Forschungsgemeinschaft (EL
160/3-2).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jörn Elsner, MD, Department of
Dermatology and Allergology, Hannover Medical University, Ricklinger
Str. 5, D-30449 Hannover, Germany; e-mail: jelsner{at}csi.com.
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Abstract
Introduction