* Division of Hematology, Institute of Clinical Medicine, University of Tsukuba
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd.
College of Medical Technology, University of Tsukuba, Japan
Correspondence: Haruhiko Ninomiya, M.D., Ph.D., College of Medical Technology, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan. E-mail: hninomiya{at}itan.tsukuba.ac.jp
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Key Words: hypereosinophilic syndrome • cytosolic phospholipase A2 • arachidonic acid
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CD69 is a type II transmembrane glycoprotein with a C-type lectin
binding domain in the extracellular portion of the molecule
[5
]. CD69 is induced in vitro on cells of
most hematopoietic lineages, including T and B lymphocytes, natural
killer (NK) cells, neutrophils, and eosinophils, whereas it is
constitutively expressed on monocytes and platelets [6
].
Human eosinophils strongly express CD69 by in vitro
stimulation with phorbol myristate acetate (PMA) and eosinophilopoietic
cytokines such as granulocyte-macrophage colony-stimulating factor
(GM-CSF), interleukin (IL)-3 and IL-5, whereas only weakly with
interferon (IFN)-
, IL-4, IL-13, type IIA phospholipase
A2 (PLA2-IIA) and lysophosphatidic acid
[4
, 7
8
9
10
]. However, it has also been
reported that eosinophils are not induced to express CD69 by
granulocyte colony-stimulating factor (G-CSF), IL-2, IL-6, and PAF
[4
, 7
]. Some of the mechanisms by which
CD69 is induced have been clarified in T lymphocytes and NK cells
[6
, 11
]. In stimulation by PMA, the signal
transduction via a protein kinase C (PKC)/Ras/Raf-1 pathway has been
revealed [12
, 13
]. Engagement of T cell
receptors on T cells or IL-2 receptor on NK cells also results in Ras
activation. In eosinophils, PMA should induce CD69 also via the
PKC/Ras/Raf-1 pathway, but the pathway initiated by other physiological
stimulants has been unclear.
Although normal peripheral blood eosinophils do not appear to express CD69, CD69 expression on peripheral blood eosinophils of hypereosinophilic syndrome (HES), although not in all cases of HES, has been shown [14 , 15 ]. While CD69 expression has been demonstrated on activated eosinophils in local sites of various diseases, it has rarely been demonstrated on peripheral blood eosinophils except for HES eosinophils. We previously reported that both cytosolic PLA2 (cPLA2) and 5-lipoxygenase (5-LO) were increased and that cPLA2, in particular, was activated and translocated to the membranes in HES eosinophils in vivo [16 ]. In a recent study, we found CD69 expression on HES eosinophils without in vitro stimulation [10 ].
In this study, we found strong CD69 induction on peripheral blood eosinophils from HES patients by in vitro PAF stimulation. This suggests that this difference in the responses to stimuli between HES and normal eosinophils could help to elucidate the signal transduction pathways for CD69 induction in eosinophils. We thus examined the effects of enzymes involved in the metabolism of arachidonic acid on PAF- and IL-5-induced CD69 expression and found a pivotal role of 5-LO in the pathway resulting in CD69 induction on blood eosinophils.
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Preparation of eosinophils
Eosinophil isolation was performed by a magnetic cell separation
system (MACS; Becton Dickinson, San Jose, CA). Heparinized (10 U/mL)
blood was drawn by venipuncture from two patients with HES and seven
normal volunteers. Informed consent was obtained from the volunteers
and HES patients after receiving appropriate information regarding the
purpose and methods of this study. The HES patients (KS and YK)
exhibited chronic (5 years and 2 years of disease duration,
respectively) eosinophilia (eosinophils, 10–40 x
109/L) without any underlying allergic disorders. The
eosinophil-rich fraction was separated as CD16-negative fraction
according to the method of Hansel et al. [18
]. The
purity of eosinophils was always more than 95%, as assessed by
staining with Diff-Quik (Kokusaishiyaku, Kobe, Japan). Cells were kept
on ice until use.
Flow cytometry for assessment of CD69 expression
Expression of CD69 on human eosinophils was measured as follows.
Cell suspensions of 2 x 106/mL were stimulated with
PAF or other stimulants at 37°C for the indicated durations.
Inhibitors were added and preincubated with eosinophils (37°C, 10
min) before the addition of stimulants. The stimulation was terminated
by placing the suspensions on ice and removing the supernatants
immediately after slight centrifugation (4°C, 5 min). The cells
resuspended in flow cytometry (FCM) buffer composed of 137 mM NaCl, 2.7
mM KCl, 8.1 mM Na2HPO4, 1.5 mM
KH2PO4, pH 7.4, containing 0.1% bovine serum
albumin and 0.02% NaN3, were incubated with 8 µg/mL
anti-CD69 Ab on ice for 30 min. After washing the cells once with FCM
buffer, the cells were reacted with FITC-labeled goat anti-mouse Ig.
Then, the cells were analyzed using a flow cytometer
(EPICS® XL-MCL, Coulter, Miami, FL). Eosinophils were
gated on the basis of their forward- and side-light scatters and cell
debris was excluded from the analysis.
The mean fluorescence intensities (MFI) in the eosinophils were obtained from the histograms of a population of at least 5,000 cells. The specific MFI for each population was determined by subtracting the nonspecific MFI from the MFI when stained with anti-CD69.
Western immunoblotting
Cytosol and membrane fractions of eosinophils were prepared
according to the previously described method [16
].
Briefly, eosinophils suspended at 2 x 107/mL in
Buffer A (80 mM KCl, 1 mM EDTA, 1 mM EGTA, 40 mg/mL leupeptin, 25 mg/mL
pepstatin A, 0.2 mM pefablock, 10 mM NaF, 0.2 mM
NH4VO3, 4 mM dithiothreitol in 10 mM HEPES, pH
7.4) were incubated on ice for 30 min. The cells were then sonicated
and centrifuged at 1,000 g at 4°C to remove debris. The
supernatant was centrifuged at 180,000 g at 4°C for 30
min, and then the recovered supernatant (cytosol fraction) and pellet
were collected. The pellet resuspended in Buffer A followed by
sonication was collected as the membrane fraction. The protein amounts
of the samples applied to sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis (PAGE) were quantified using a Bio-Rad Protein
Assay (Bio-Rad Laboratories, Hercules, CA). Each sample (25 µg of
protein) was electrophoresed on an SDS-PAGE of 4–20% gradient
polyacrylamide. The proteins were then transferred to an Immobilon-P
membrane (Millipore, Bedford, MA) and probed with mouse monoclonal
anti-5-LO Ab (0.5 µg/mL). Signals on the membrane were developed by
enhanced chemiluminescence (ECL) using horseradish
peroxidase-conjugated anti-mouse Ig.
Cell viability
Cell viability assessed by trypan blue exclusion was always
greater than 90% even in the highest concentration of inhibitor.
Statistical analysis
Statistical analysis was performed using Student’s unpaired
t test for the comparisons between two groups. Statistical
significance was assumed when P < 0.05 vs. control.
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Figure 1. Comparison of CD69 induction by PAF or IL-5 between on HES and normal
eosinophils. Eosinophils from HES patients and normal donors were
incubated at 37°C for 3.5 h with 100 nM PAF or 50 ng/mL IL-5.
(A) Representative histograms of the fluorescence intensity of CD69
expression (bold line) on eosinophils after incubation. The
fluorescence intensity of eosinophils stained with the isotype control
Ab is also shown (thin line). As for HES eosinophils, the experiments
were repeated at least three times every 4 weeks and similar findings
were obtained each time. (B) Values are expressed as the mean ±
SD of specific MFI measured in two independent experiments
on two HES patients (HES #1 and HES #2) and five independent
experiments on five normal donors. Each assay was performed in
triplicate.
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Figure 2. Dose-dependency of CD69 induction by PAF on both eosinophils from HES
patients and normal donors. Eosinophils from HES patients and normal
donors were incubated in the presence of various concentrations of PAF
for 3.5 h at 37°C. Eosinophils were stained with anti-CD69 Ab,
and its bindings to the cells were measured using flow cytometry.
Induction of CD69 expression is indicated as the mean of specific MFI
(arbitrary units) measured in two independent experiments on two HES
patients (open circles and squares) or as the mean ±
SD of specific MFI measured in five independent experiments
on five normal donors (filled circles). Each assay was performed in
triplicate.
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Figure 3. Effects of various inhibitors on PAF-induced CD69 expression on HES
eosinophils. Eosinophils from HES patients were pretreated with YM264,
AACOCF3, AA861, MK-886, and indomethacin for 10 min at
37°C. The eosinophils were then incubated with 100 nM PAF for
3.5 h. Induction of CD69 expression is indicated as the mean of
two independent experiments (in triplicate) on two HES patients.
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Figure 4. Effects of various inhibitors on IL-5-induced CD69 expression on human
eosinophils. Eosinophils from HES patients or normal donors were
preincubated with AA861, MK-886, AACOCF3, and PD98059 for
10 min at 37°C. The eosinophils were then incubated with 50 ng/mL
IL-5 for 3.5 h. Induction of CD69 expression is indicated as the
mean of two independent experiments on two HES patients (A) and as
mean ± SD of five independent experiments on five
normal donors (B). Each assay was performed in triplicate.
*P < 0.05 compared with the buffer control.
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Translocation of 5-LO on human eosinophils after treatment with
IL-5 or PAF
In activated leukocytes (neutrophils and monocytes), 5-LO is
translocated from the cytosol predominantly to the nuclear envelope,
where it is localized in close proximity to its activating protein
(5-lipoxygenase-activating protein, FLAP) and cPLA2
[19
]. As shown in Figure 5
, 5-LO was translocated from the cytosol to the membranes after a
1-h incubation in the presence of either IL-5 (50 ng/mL) or PAF (100
nM) in eosinophils from both HES patients and normal donors.
 View larger version (21K): [in a new window] |
Figure 5. Western immunoblot of 5-LO in human eosinophils. Eosinophils from
patients with HES and normal donors were incubated with or without 50
ng/mL IL-5 or 100 nM PAF in the presence of 1.3 mM Ca2+
(37°C, 1 h). The cells were then fractionated into cytosol and
membrane fractions as described in Materilas and Methods. The fractions
(25 µg of protein) were subjected to SDS-PAGE (4–20%
polyacrylamide, reduced condition) followed by immunoblotting with
anti-5-LO. The values of HES patients are from one representative
experiment of two patients. The values of normal donors are from one
representative experiment of three experiments.
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Figure 6. Augmentation of CD69 expression by PAF in combination with
arachidonic acid. Eosinophils were incubated with various
concentrations of PAF in the absence (filled circles) or presence
(filled squares) of 1 µM arachidonic acid for 3.5 h at 37°C.
Induction of CD69 expression is indicated as the mean ±
SD of five independent experiments on five normal donors.
Each assay was performed in triplicate. *P < 0.05
compared with the buffer control.
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Figure 7. Induction of CD69 expression by 5-LO pathway products
on human eosinophils. (A) Eosinophils from HES patients and normal
donors were incubated at 37°C for 3.5 h with 1 µM 5-oxo-ETE.
Representative histograms of the fluorescence intensity of eosinophils
stained with anti-CD69 Ab (bold line) or control Ab (thin line).
5-oxo-ETE induced intense CD69 expression on both HES and normal
eosinophils. (B) Eosinophils from HES patients and normal donors were
incubated at 37°C for 3.5 h with 1 µM 5-LO pathway products
(5-HPETE, 5-HETE, 5-oxo-ETE, LTB4, LTC4,
LTD4, or LTE4). Induction of CD69 is expressed
as the mean of specific MFI in two independent experiments on two HES
patients (left) or as mean ± SD of five independent
experiments on five normal donors (right). Each assay was performed in
triplicate. *P < 0.05 compared with the buffer
control.
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It was unclear whether 5-LO was involved in the CD69 induction on eosinophils by physiological stimulants other than PAF. IL-5-induced CD69 expression on blood cells is unique to eosinophils because the specific receptor for IL-5 is absent on neutrophils and rarely, if ever, expressed on other cells that express CD69 [22 , 23 ]. As shown in Figure 1 , IL-5 markedly induced CD69 expression on eosinophils from both HES patients and normal donors. Both AA861 and MK-886 inhibited IL-5-induced CD69 expression dose-dependently in eosinophils from both HES patients and normal donors (Fig. 4) , as well as in the case of PAF-induced CD69 expression on HES eosinophils. We also examined the effects of AA-861 (10 µM) on IL-3 (5 ng/mL)- or GM-CSF (5 ng/mL)-induced CD69 expression on eosinophils from both HES patients and normal donors; similar findings to those in the case of IL-5 were obtained (data not shown). These findings suggest a critical role of 5-LO in IL-5-induced CD69 expression on both eosinophils. Although AACOCF3 did not inhibit PAF-induced CD69 expression, AACOCF3 and PD98059 inhibited IL-5-induced CD69 expression on eosinophils from normal donors, but hardly on those from HES patients (Fig. 4) . PD98059 is known to inhibit cPLA2 activation, partially because phosphorylation of cPLA2 by MAP kinase increases its enzymatic activity [24 , 25 ]. These findings suggest that there are differences in the MAP kinase activation and the subsequent cPLA2 activation for the CD69 induction on eosinophils between HES patients and normal donors.
5-LO is activated by its translocation from the cytosol to the nuclear membranes and its interaction with FLAP, and this process is regulated by the level of cytosolic Ca2+ [26 ]. The findings that IL-5- and PAF-induced translocation of 5-LO in both eosinophil types (Fig. 5) indicated that IL-5 and PAF activate 5-LO. Moreover, exogenously added arachidonic acid markedly enhanced PAF-induced CD69 expression on normal eosinophils (Fig. 6) . These findings strongly suggest that 5-LO activation is required for CD69 induction in both HES and normal eosinophils, but cPLA2 activation, supplying arachidonic acid, is required for induction on normal eosinophils. cPLA2 is also activated by Ca2+-dependent translocation from the cytosol to the nuclear membranes, similar to the case of 5-LO, and by phosphorylation on its serine residues [24 , 27 ]. Human eosinophils generate 5-LO metabolites in only small amounts after stimulation with receptor agonists such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and PAF, but in high yields after stimulation with Ca2+ ionophore [28 , 29 ]. Schatz-Munding et al. explain this phenomenon in polymorphonuclear leukocytes as follows: release of Ca2+ from the intracellular pool by receptor agonists is sufficient for 5-LO activation but is insufficient for cPLA2 activation to supply arachidonic acid [30 ]. In normal eosinophils, stimulation with PAF would activate 5-LO but would not generate sufficient 5-LO products because of the shortage of 5-LO substrate, arachidonic acid, which is produced by cPLA2, whereas stimulation with IL-5 would activate both cPLA2 and 5-LO, resulting in sufficient generation of 5-LO products [31 , 32 ]. However, IL-5 may induce translocation of 5-LO in a different way from PAF, for it has been reported that IL-5 by itself hardly induces an increase in intracellular Ca2+ [33 , 34 ]. It has been demonstrated that translocation of 5-LO is a calcium-dependent process [26 ], and in addition, the translocation of 5-LO requires its phosphorylation by protein tyrosine kinases [35 , 36 ]. Taken together with the previous studies [32 ], although it has not been determined yet which tyrosine kinase-dependent pathway is involved in the IL-5-induced 5-LO translocation, IL-5 may induce a tyrosine kinase-dependent redistribution of 5-LO to the eosinophil nucleus. Meanwhile, lipid bodies are the sites of esterified arachidonate localization in diverse cell types, including eosinophils, and they may play a major role in the formation of eicosanoid mediators during inflammation [37 , 38 ]. In eosinophils, increased numbers of lipid bodies have been demonstrated in patients with HES [38 ]. Abundant lipid bodies are possibly substituted for novel arachidonate production by cPLA2 in HES eosinophils. Moreover, 5-LO was shown within eosinophil lipid bodies [39 , 40 ]. Therefore, the lipid bodies may be the major sites containing substrate for 5-LO in HES eosinophils.
Because 5-LO plays a pivotal role in CD69 induction on human eosinophils, some of its enzymatic products may be involved in the induction of CD69 on human eosinophils. The enzymatic products of 5-LO are classified into two groups: LTs (LTA4, LTB4, LTC4, LTD4, and LTE4) and eicosatetraenoic acids (5-HPETE, 5-HETE, and 5-oxo-ETE). Whereas LTs did not induce CD69 expression on human eosinophils, all the eicosatetraenoic acids examined induced CD69 expression (Fig. 7) . Among the substances at the same concentration, 5-oxo-ETE induced CD69 most intensely on eosinophils from both HES patients and normal donors. 5-oxo-ETE is known to be a potent stimulator of many eosinophil functions, e.g., migration, L-selectin shedding, surface expression of CD11b, actin polymerization, and calcium mobilization in cells [41 , 42 ]. The finding of this study is the first demonstration of the effect of 5-oxo-ETE on CD69 expression on human eosinophils. Because eosinophils can synthesize 5-oxo-ETE from arachidonic acid in addition to 5-HPETE and 5-HETE [42 ], this suggests that endogenous eicosatetraenoic acids (5-HPETE, 5-HETE, and 5-oxo-ETE) are strongly involved in the induction of CD69 expression on human eosinophils.
In conclusion, we demonstrated a critical role of 5-LO in CD69 induction by in vitro stimulation such as PAF or IL-5 on human blood eosinophils. A major contribution of a pathway other than cPLA2, which supplies arachidonic acids, was suggested to be involved in the strong CD69 induction in HES eosinophils. It was suggested that CD69 induction in eosinophils was induced via arachidonic acid metabolites such as 5-HPETE, 5-HETE, and 5-oxo-ETE.
Received June 12, 2000; revised August 21, 2000; accepted August 22, 2000.
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