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(Journal of Leukocyte Biology. 2001;69:105-112.)
© 2001 by Society for Leukocyte Biology

Pivotal role of 5-lipoxygenase in the activation of human eosinophils: platelet-activating factor and interleukin-5 induce CD69 on eosinophils through the 5-lipoxygenase pathway

Tetsuya Urasaki*, Jun Takasaki{dagger}, Toshiro Nagasawa* and Haruhiko Ninomiya{ddagger}

* Division of Hematology, Institute of Clinical Medicine, University of Tsukuba
{dagger} Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd.
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD69 is an activation-related cell surface molecule on human eosinophils. It has been reported that interleukin (IL)-5, but not platelet-activating factor (PAF), can induce CD69 on human eosinophils in vitro. In this study, PAF induced CD69 intensely on eosinophils from patients with hypereosinophilic syndrome (HES), while only weakly on those from normal donors. Because HES eosinophils contain abundant cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO), we examined the roles of several enzymes involved in the metabolism of arachidonic acid in the PAF- or IL-5-induced CD69 expression on eosinophils. The CD69 expression induced by PAF and IL-5 on HES eosinophils and that by IL-5 on normal eosinophils were both inhibited by AA861 and MK-886, inhibitors of 5-LO activity. In addition, AACOCF3, a selective cPLA2 inhibitor, inhibited IL-5-induced CD69 expression on normal eosinophils, although it hardly affected either IL-5- or PAF-induced CD69 expression on HES eosinophils. Moreover, PAF alone induced CD69 only weakly on normal eosinophils, but exogenous arachidonic acid remarkably enhanced PAF-induced CD69 expression on them. These findings suggest that IL-5 activates both cPLA2 and 5-LO but PAF activates only 5-LO. It is suggested that 5-LO plays a critical role in the induction of CD69 on eosinophils.

Key Words: hypereosinophilic syndrome • cytosolic phospholipase A2 • arachidonic acid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophils are major effector cells contributing to inflammation in allergic disorders such as asthma and atopic dermatitis [1 , 2 ]. They are activated by diverse stimuli and release various proinflammatory mediators, i.e. eosinophil-derived granular basic proteins such as major basic protein, eosinophil cationic protein, and eosinophil peroxidase, lipid mediators such as platelet-activating factor (PAF) and leukotriene (LT) C4, reactive oxygen species, and various cytokines [3 ]. The finding that CD69 expression was observed on activated eosinophils at inflamed sites but not on peripheral blood eosinophils suggests that CD69 may be a useful marker for activated eosinophils [4 ].

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)-{alpha}, 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
PAF (C18) was obtained from Bachem (Bubendorf, Switzerland); recombinant human (rh) IL-5 and rhIL-3 were obtained from Calbiochem (La Jolla, CA); rhGM-CSF was obtained from Genzyme (Cambridge, MA); AA861, indomethacin, arachidonic acid, LTB4, LTC4, LTD4, and LTE4 were obtained from Sigma (St. Louis, MO); AACOCF3, MK-886, and 5-HPETE were obtained from Biomol Research Laboratories (Plymouth Meeting, PA); PD98059 was obtained from Research Biochemicals International (Natick, MA); YM264, a PAF inhibitor, was chemically synthesized using methods described previously [17 ]; 5-HETE and 5-oxo-ETE were obtained from Cayman Chemical (Ann Arbor, MI); monoclonal anti-CD69 antibody (Ab) was obtained from Immunotech (Marseille, France); mouse immunoglobulin (Ig) G was obtained from Cappel (Turnhout, Belgium); fluorescein isothiocyanate (FITC)-labeled goat F(ab’)2 anti-mouse Ig and horseradish peroxidase-conjugated goat F(ab’)2 anti-mouse Ig were obtained from BioSource International (Camarillo, CA); monoclonal anti-5-LO Ab was obtained from Transduction Laboratories (Lexington, KY).

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of CD69 induction on HES and normal eosinophils by PAF and IL-5
We examined the effects of PAF and IL-5 on CD69 expression on human peripheral blood eosinophils from HES patients and normal donors. It has been reported that IL-5 and PAF are positive and negative inducers for CD69 on eosinophils, respectively. PAF markedly induced CD69 expression on HES eosinophils, but not on normal eosinophils (Fig. 1 ). In contrast to the findings of PAF, IL-5 markedly induced CD69 expression on both HES and normal eosinophils (Fig. 1) . The IL-5-induced CD69 expression reached a plateau level at 50 µg/mL (data not shown). It has been reported that stimulation of eosinophils from donors with mild eosinophilia with PAF (1 µM) for up to 2 h did not induce CD69 expression [7 ]. As shown in Figure 2 , the level of CD69 expression on HES eosinophils by PAF was much higher than that on normal eosinophils at various concentrations of PAF. The intensity of CD69 expression induced by PAF on normal eosinophils was very weak.



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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.

 
Involvement of 5-LO in PAF-induced CD69 expression on HES eosinophils
We previously reported that both cPLA2 and 5-LO were increased and that cPLA2 was already activated and translocated to the membranes in eosinophils of HES patients in vivo [16 ]. To verify the possibility that an increased level of cPLA2 and 5-LO was involved in the intense CD69 induction on HES eosinophils, we examined the contributions of pivotal enzymes involved in the metabolism of arachidonic acid, i.e., cPLA2, 5-LO, and cyclooxygenases (COXs). We first confirmed the effect of PAF on CD69 induction on HES eosinophils by its inhibition by a PAF inhibitor, YM264, in a dose-dependent manner (Fig. 3 ). Then, the effects of various inhibitors on PAF-induced CD69 expression on HES eosinophils were examined. Both AA861 and MK-886, inhibitors of 5-LO activity, inhibited PAF-induced CD69 expression on HES eosinophils in dose-dependent manners, whereas AACOCF3, a cPLA2 inhibitor, and indomethacin, a COX inhibitor, did not (Fig. 3) . Because both MK-886 and AACOCF3, at a concentration of 10 µM, induced signals to a high extent, which seemed to be nonspecific, we omitted these data from Figure 3 and Figure 4 . In addition, any inhibitors we used in this study did not affect the results of nonstimulated eosinophils.



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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.

 
Involvement of 5-LO and cPLA2 in IL-5-induced CD69 expression on human eosinophils
In contrast to the case of PAF, IL-5 markedly induced CD69 expression on both HES and normal eosinophils (Fig. 1) . We then examined the effects of various inhibitors of arachidonate metabolite-forming enzymes on IL-5-induced CD69 expression on both HES and normal eosinophils. Both inhibitors of 5-LO activity, AA861 and MK-886, inhibited IL-5-induced CD69 expression on eosinophils from both HES patients (Fig. 4A) and normal donors (Fig. 4B) , and AACOCF3 hardly influenced the IL-5-induced CD69 expression on HES eosinophils, as in the case of PAF-induced CD69 expression (Fig. 3) . However, AACOCF3 inhibited IL-5-induced CD69 expression on normal eosinophils in a dose-dependent manner (Fig. 4B) . Moreover, PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase, hardly inhibited IL-5-induced CD69 expression on HES eosinophils (Fig. 4A) , but did on normal eosinophils (Fig. 4B) . These findings suggest that the activity of cPLA2, activated by MAP kinase, is involved in the IL-5-induced CD69 expression on normal human eosinophils but not necessarily for that on HES eosinophils.

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.



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  		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.

 
Effects of arachidonic acid and 5-LO products on CD69 induction on human eosinophils
If the difference of PAF-induced CD69 expression between HES and normal eosinophils (Fig. 2) was caused by the state of cPLA2, arachidonic acid, a predominant enzymatic product by cPLA2, could enhance the PAF-induced CD69 expression on eosinophils from normal donors. Accordingly, we examined the effect of arachidonic acid on PAF-induced CD69 expression on eosinophils from normal donors. Arachidonic acid (1 µM) significantly enhanced PAF (100 nM and 1 µM)-induced CD69 expression on eosinophils from normal donors, whereas arachidonic acid (1 µM) or PAF (100 nM and 1 µM) alone barely induced CD69 expression on eosinophils from normal donors (Fig. 6 ).



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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.

 
5-LO and FLAP are both required for cellular LT synthesis, with 5-LO catalyzing both the synthesis of 5-HPETE from arachidonic acid and the subsequent synthesis of LTB4, LTC4, LTD4, and LTE4 [20 ]. Moreover, 5(S)-hydroperoxyeicosatetraenoic acid (5-HPETE) is converted to 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) by specific enzymes [20 , 21 ]. Thus, we examined the effects of LTs (LTB4, LTC4, LTD4, and LTE4) and the other 5-LO products (5-HPETE, 5-HETE, and 5-oxo-ETE) on CD69 induction on blood eosinophils from HES patients and normal donors. As shown in Figure 7 , 5-HPETE, 5-HETE, and 5-oxo-ETE induced CD69 expression on eosinophils from both types of donors. Among these substances, 5-oxo-ETE appeared to induce CD69 expression most intensely. No significant induction of CD69 expression was observed by LTs except by LTB4, which slightly induced CD69 expression on HES eosinophils.



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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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Hartnell et al. reported that stimulation of eosinophils with PAF (1 µM) for up to 2 h did not induce CD69 expression [7 ]. No other studies concerning the effects of PAF on the CD69 induction on eosinophils have been available. As shown in Figure 1 , PAF markedly induced CD69 on HES eosinophils in comparison with normal eosinophils. When the dose-dependency of the effect of PAF on the CD69 induction on human eosinophils from normal donors was examined, the CD69 induction was barely detected after a 3.5-h incubation at 37°C (Fig. 2) . We previously reported that both cPLA2 and 5-LO, involved in the liberation or metabolism of arachidonate, were increased and cPLA2, in particular, was already activated and translocated to the membranes of eosinophils of HES patients in vivo [16 ]. AA861 inhibits 5-LO competitively, whereas the inhibition of 5-LO by MK-886 is mediated by its interaction with FLAP. AA861 and MK-886 inhibited PAF-induced CD69 expression on HES eosinophils in dose-dependent manners (Fig. 3) . However, in the case of HES eosinophils, AACOCF3 and indomethacin did not inhibit it at all. These findings suggest a critical role of 5-LO in PAF-induced CD69 expression on HES eosinophils.

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.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr. Masao Katoh (Yamanouchi Pharmaceutical Co.) for support throughout this work and to Dr. Toshimitsu Yamada (Yamanouchi Pharmaceutical Co.) for generously donating YM264.

Received June 12, 2000; revised August 21, 2000; accepted August 22, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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