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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 601-609
IMMUNOBIOLOGY
From the Departments of Pediatrics and Microbiology, Saga Medical
School, Saga, Japan.
Interleukin (IL)-4, IL-10, and IL-13, Th2 cell-derived cytokines,
play major roles in the pathophysiology of allergic diseases. These
cytokines up-regulate or down-regulate the production of arachidonic
acid metabolites. In this study, we have investigated the effect of
IL-4, IL-10, IL-13, and other cytokines on A23187-stimulated synthesis
of leukotriene (LT) B4 in human polymorphonuclear
leukocytes (PMNs). Production of LTB4 was measured by
specific radioimmunoassay and high performance liquid chromatography.
Messenger RNA (mRNA) expression of cytosolic phospholipase
A2 (cPLA2), 5-lipoxygenase (5-LO), and
LTA4 hydrolase, which were involved in the synthesis of
LTB4, was determined by reverse transcription-polymerase
chain reaction and Northern blot analysis. Protein synthesis of
their enzymes was determined by Western blot analysis.
IL-4 and IL-13 enhanced A23187-stimulated LTB4 synthesis
and increased mRNA expression and protein synthesis of LTA4
hydrolase, but not those of cPLA2 or 5-LO.
These results indicate that IL-4 and IL-13 transcriptionally or
post-transcriptionally up-regulate the synthesis of LTB4, a potent chemotactic factor to PMNs, at the enzyme level of
LTA4 hydrolase, and this up-regulation mechanism may
participate in the development of allergic inflammation.
(Blood. 2000;96:601-609)
Cytokines have been recognized as important bioactive
substances produced in lymphocytes and various other cell types. It is
accepted that they form a network with one another and regulate functions of other class mediators, such as arachidonic acid (AA) metabolites. Interleukin (IL)-4, IL-10, and IL-13, which are CD4 helper type 2 lymphocyte (Th2 cell)-derived cytokines, play major roles in the pathophysiology of allergic diseases. Th2 cell-derived cytokines potentiate mucosal inflammation and bronchial hyperreactivity in asthma patients.1-4 It was also reported that inhalation
of IL-4 and IL-5 increased the number of eosinophils and neutrophils in
bronchoalveolar lavage fluid (BALF) in human beings.5,6
In asthmatic patients, the number of polymorphonuclear leukocytes
(PMNs) were increased in BALF after allergen challenge; neutrophils
appeared first; then eosinophils replaced them.7-9 Although
various kinds of chemoattractants are involved in the infiltration of
PMNs, leukotriene (LT) B4 is one of the most potent chemotactic factors affecting PMNs. Neutrophils themselves are also a
major source of LTB4 as well as prostaglandin (PG)
E2 and thromboxane (TX) A2. Thus,
LTB4 produced by neutrophils is possibly involved in the
process of allergic inflammation. LTB4 is an AA metabolite
of 5-lipoxygenase (5-LO) pathway and is synthesized by 3 consecutive
enzymatic reactions of cytosolic phospholipase A2
(cPLA2), 5-LO, and LTA4 hydrolase.
Recent studies demonstrated that the production of AA metabolites was
enhanced by stimulation with various bioactive substances such as
interleukins in various cell types, including PMNs. One mechanism of
the enhanced production is a new induction of synthesizing enzymes such
as cPLA2, cyclooxygenase (COX)-2, and 5-LO by
transcriptional and/or post-transcriptional regulation of
genes.10-19 We and others recently demonstrated that
production of thromboxane and prostaglandin was transcriptionally
up-regulated by lipopolysaccharide (LPS) at the enzyme levels of
cPLA2 and COX-2 in PMNs.20-22 However, there
have been few reports on whether similar regulatory mechanisms are
present in the 5-LO pathway in PMNs. In this study, we have examined
whether various cytokines, especially allergy-related Th2 cytokines,
stimulate synthesis of LTB4, the final product of the 5-LO
pathway in PMNs, and have further investigated the mechanisms of this regulation.
Reagents
Isolation and culture of human PMNs
Radioimmunoassay for LTB4 and TXB2 PMNs were incubated for various periods of time with various concentrations of IL-4, IL-10, IL-13, IL-6, IL-2, or LPS as described above. After incubation and washing with PBS, the cells (1 × 105 cells) were resuspended in 1 mL of RPMI medium without serum and stimulated with A23187 at 10 5 mol/L for 15 minutes at 37°C under 5%
CO2 in air. The medium was collected for measurement of
LTB4 and TXB2 by specific radioimmunoassay (RIA). RIA for LTB4 was performed as previously
described.18 In brief, 100 µL
of sample was incubated at 4°C for 10 hours with 100
µL each of anti-LTB4 rabbit serum,
[3H]-LTB4, and 50 mmol/L sodium phosphate
buffer containing 0.9% NaCl and gamma globulin. After incubation, free
[3H]-LTB4 was separated by centrifugation
after the addition of 500 µL of gamma globulin-coated
charcoal (Norit A) to the assay mixture. Radioactivity of the
supernatant was measured with a liquid scintillation counter, Model LS
7500 (Beckman; Irvine, CA). The amount of LTB4 was
calculated from a standard curve. The detectable range of
LTB4 was from 16 to 100 pg/tube. RIA for TXB2
was performed under the same conditions except that
anti-TXB2 rabbit serum and
[3H]-TXB2 were used instead of
anti-LTB4 rabbit serum and
[3H]-LTB4, respectively. The detectable range
of TXB2 was from 32 to 1000 pg/tube.
Measurement of 5-LO products of AA metabolism by high performance liquid chromatography Measurement of LTB4, 5-hydroxyeicosatetraenoic acid (5-HETE), and 6-trans LTB4 diastereoisomers by high performance liquid chromatography (HPLC) was performed as previously described.23 PMNs were incubated for various periods of time with various concentrations of IL-4, IL-10, and IL-13 as described above. After incubation and washing with PBS, the cells were resuspended in RPMI at 2 × 106 cells/mL and stimulated with A23187 at 105 mol/L for 15 minutes.
The medium was then removed and mixed with 3 mL methanol. The methanol
samples were centrifuged at 3000 rpm for 5 minutes, and the
supernatants were partially purified and concentrated by passing
through a Sep Pak C18 cartridge (Waters; Milford, MA) and applied to
HPLC system, model TOSO-8010 (Toso; Tokyo, Japan), equipped with a Nova
Pak C18 column (Waters). The sample was delivered with acetonitorile,
methanol, water, and acetic acid (31:17.68:51.24:0.68 [vol/vol]) for
the assay of LTB4 and 6-trans LTB4
diastereoisomers and (45:13.62:40.84:0.54 [vol/vol]) for the assay of 5-HETE, respectively. The flow rate was 0.8 mL/min, and the absorbance was monitored by a spectrophotometer at 280 nm
for LTB4 and 6-trans LTB4 diastereoisomers, and
at 235 nm for 5-HETE. Quantification of LTB4, 6-trans
LTB4 diastereoisomers, and 5-HETE was based on the peak
ratio area-to-standards.
Analysis of messenger RNA expression for cPLA2, 5-LO, FLAP, and LTA4 hydrolase by reverse transcription-PCR PMNs were incubated with IL-4, IL-10, IL-13, IL-6, IL-2, or LPS as described above. After incubation, the cells were washed with PBS and obtained (5 × 106 cells). RNA extraction from the cells was performed as previously described.18 The cells were lysed with 1 mL of isogen. After adding chloroform (200 µL) and centrifuging at 12 000g for 15 minutes at 4°C, we collected the aqueous phase. The sample was again centrifuged at 12 000g for 15 minutes at 4°C after the addition of isopropanol (500 µL): the supernatant was discarded. After the addition of 75% ethanol (1 mL) to the pellet, the sample was centrifuged at 12 000g for 5 minutes at 4°C. The supernatant was again discarded, and the pellet containing total RNA was dried under room air. The extracted total RNA was dissolved in DEPC-treated water and quantified by measurement of absorbance at 260 nm with a spectrophotometer, model DU-600 (Beckman).Northern blot analysis for LTA4 hydrolase PMNs were incubated with IL-4 (10 ng/mL), IL-10 (100 ng/mL), IL-13 (100 ng/mL), or LPS (100 ng/mL) for 6 hours. After incubation and washing with PBS, the cells (5 × 107 cells) were obtained, and total RNA was isolated. Northern blot analysis was performed as previously described.24 Fifteen micrograms of total RNA was denatured at 65°C for 5 minutes in 50% formamide, electrophoresed through 1% agarose gel containing 6.6% formaldehyde, and transferred to a nylon membrane. A DNA fragment (216 bp) was prepared by RT-PCR with the use of a primer pair for LTA4 hydrolase. The DNA fragment was labeled with [32P]
dCTP by means of a random primer labeling kit and used as a DNA probe
for LTA4 hydrolase mRNA. Hybridization was done overnight at 60°C in 5 × saline sodium citrate containing 4 × Denhardt's solution, 1% sodium dodecyl sulfate (SDS), 50 mmol/L
sodium phosphate, and 0.1 mg/mL of denatured salmon sperm DNA. The
membrane was washed with 2 × SSC, 0.1% SDS at room temperature
(4 times, 10 minutes each time), followed by 0.2 × SSC, 0.1% SDS
at 60°C (twice, 30 minutes each time). The hybridized bands were
visualized by autoradiography. The same membrane was rehybridized with
a human beta-actin probe as a control.
Western blot analysis for enzyme proteins of cPLA2, 5-LO, and LTA4 hydrolase PMNs were incubated for various periods of time with various concentrations of IL-4, IL-10, IL-13, IL-6, IL-2, or LPS as described above. Western blot analysis for cPLA2 protein was performed as previously described.20 In brief, after incubation and washing with PBS, the cells (1 × 107 cells) were obtained and lysed in 100 µL of solubulization buffer containing 1% Tween 20, 1 mmol/L phenylmethylsulfonyl fluoride, and 50 mmol/L Tris-HCl (pH 8.0) on ice. The cells were sonicated by means of a sonicator, model R-225R (Heat-Systems-Ultrasonics Inc; Plainview, NY), on ice 3 times for 5 seconds and centrifuged at 12 000g for 20 minutes. The supernatant was collected, and the protein concentration was determined by Lowry's method. The samples (20 µg each as protein amount) were separated by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto nitrocellulose membrane. The membrane was incubated with blocking solution Block Ace (Dainihon Pharmaceutical; Tokyo, Japan) for 1 hour at room temperature, and then incubated with antihuman cPLA2 mouse monoclonal antibody diluted at 1:500 with the diluent solution containing 10% blocking buffer at room temperature for 1 hour. The membrane was incubated with HRP-conjugated antimouse IgG goat serum diluted at 1:10 000 with the diluent solution at room temperature for 1 hour. The enzyme protein of cPLA2 was visualized by the use of an enhanced chemiluminescence system, Supersignal Substrate Western Blotting (Pierce; Rockford, IL), and photographed by a luminescent image analyzer, LAS 1000 (Fuji Film; Tokyo, Japan). Western blot analysis for enzyme proteins for 5-LO and LTA4 hydrolase was performed under the same conditions except that antihuman 5-LO mouse polyclonal antibody diluted at 1:1000 and HRP-conjugated antimouse IgG goat serum were used for the 5-LO assay, and antihuman LTA4 hydrolase rabbit polyclonal antibody diluted at 1:500 and HRP-conjugated antirabbit IgG goat serum were used for the LTA4 hydrolase assay instead of antihuman cPLA2 mouse monoclonal antibody and HRP-conjugated antimouse IgG goat serum.Analysis of LTA4 hydrolase activity PMNs were incubated with IL-4, IL-10, IL-13, IL-6, IL-2, or LPS as described above. After incubation and washing, the cells (1 × 107 cells) were obtained and resuspended in 100 µL of Hepes buffer (137 mmol/L NaCl, 2.6 mmol/L KCl, 0.36 mmol/L NaH2PO4, 10 mmol/L Hepes, and 1 mmol/L EDTA at pH 7.5) containing 1 mmol/L of dithiothreitol. The cells were sonicated on ice and centrifuged at 12 000g for 20 minutes. The supernatant was collected, and the lysate was used as an enzyme source after the protein concentration was determined by Lowry's method. LTA4-free acid prepared from LTA4 methylester was diluted with 20 µL of Hepes buffer containing 10 mg/mL of bovine serum albumin and incubated with 180 µL of the cell lysate containing GSH for 5 minutes at 37°C. The reaction was stopped by adding 2 mL of methanol. The production of LTB4 for determination of LTA4 hydrolase activity was measured by HPLC as described above.Statistical analysis Data were shown as mean ± SEM. Statistical analysis was performed by 1- way analysis of variance. P < .05 was defined as statistically significant.
Effect of IL-4, IL-10, and IL-13 on A23187-stimulated production of LTB4 determined by RIA in human PMNs Cell viability in the control was 97.5 ± 0.6% (0-hour incubation time); this became 95.5 ± 0.6% after 9 hours' incubation and then decreased to 85.8 ± 1.5% (P < .05) and 73.7 ± 1.7% (P < .05) after 15 and 24 hours' incubation, respectively. IL-4, IL-10, and IL-13 had no significant effects on the cell viability after a 9-hour incubation period (data not shown). We therefore determined the effects of IL-4, IL-10, and IL-13 on LTB4 synthesis in human PMNs for up to 9 hours' incubation. LTB4 synthesis in PMNs was not detectable in the absence of A23187 stimulation with or without IL-4, IL-10, or IL-13. As shown in Figure 1A, A23187-stimulated production of LTB4 was significantly increased from 2060 ± 108.9 pg/105 cells to 5505 ± 270.2 pg/105 cells by IL-4 (10 ng/mL) and to 3930 ± 218.1 pg/105 cells by IL-13 (100 ng/mL) after 6 hours' incubation. In a dose-dependent manner, IL-4 and IL-13 increased A23187-stimulated synthesis of LTB4, whose maximal values were obtained at 10 ng/mL and 100 ng/mL, respectively (Figure 1B). IL-10 showed no significant effects on LTB4 synthesis. The values of LTB4 production after 15 and 24 hours' incubation with and without IL-4, IL-10, and IL-13 were measured in other sets of experiments, which were smaller than those after 6 and 9 hours' incubation (data not shown).
Effect of IL-4, IL-10, and IL-13 on A23187-stimulated production of LTB4, 6-trans LTB4 diastereoisomers, and 5-HETE determined by HPLC in human PMNs We also measured the production of LTB4, 6-trans LTB4 diastereoisomers, and 5-HETE by using HPLC after 6 hours' incubation with IL-4, IL-10, or IL-13. As shown in Table 1, A23187-stimulated production of LTB4 was significantly increased by the treatment with IL-4 or IL-13. However, no significant effects were shown by the treatment with IL-4, IL-10, or IL-13 in the production of 6-trans LTB4 diastereoisomers and 5-HETE.
Effect of IL-4 on mRNA expression for cPLA2, 5-LO, FLAP, and LTA4 hydrolase in human PMNs We determined mRNA expression for LTA4 hydrolase and other enzymes, cPLA2, 5-LO, and FLAP by using RT-PCR analysis. Time-course and dose-response studies treated with IL-4 are shown in Figures 2 and 3. mRNA for LTA4 hydrolase began to increase after 3 hours' incubation with IL-4 and reached its peak value after 6 hours. Dose-response study demonstrated significant enhancement of LTA4 hydrolase mRNA at a concentration higher than 1 ng/mL. mRNA expression for cPLA2, 5-LO, and FLAP was not changed by IL-4-treatment. IL-13 also increased mRNA expression for LTA4 hydrolase after 6 hours' incubation at a concentration of 100 ng/mL (Figure 7B). Northern blot analysis demonstrated that mRNA expression for LTA4 hydrolase was markedly increased after 6 hours' incubation with IL-4 and IL-13, but not after incubation with IL-10 (Figure 7C).
Effect of IL-4 on enzyme protein-synthesis of cPLA2, 5-LO, and LTA4 hydrolase in human PMNs We used Western blot analysis to determine the effects of IL-4 on the synthesis of 3 enzymes that were involved in LTB4 synthesis: cPLA2, 5-LO, and LTA4 hydrolase. As shown in Figure 4A, protein synthesis of LTA4 hydrolase was significantly increased to 2.8 ± 0.2-fold after 6 hours' incubation. The dose-response study demonstrated significant increase at a concentration higher than 1 ng/mL (Figure 4B). Densities of cPLA2 and 5-LO proteins were not changed by IL-4 treatment. Representative results of the time-course and the dose-response studies are shown in Figure 5. IL-13 also increased the amount of LTA4 hydrolase after 6 hours' incubation at the concentration of 100 ng/mL (Figure 7D).
Effect of IL-4 on LTA4 hydrolase activity in human PMNs We further examined the effect of IL-4 on LTA4 hydrolase activity by an enzyme assay using the cell lysate as an enzyme source and LTA4-free acid as the substrate. The activity of LTA4 hydrolase in PMNs without IL-4 treatment was 151.7 ± 5.8 ng/mg protein/5 min. The level of LTA4 hydrolase activity was significantly increased to 273.9 ± 4.4 ng/mg protein/5 min after incubation with IL-4 (10 ng/mL) for 6 hours (Figure 6A). Dose-response study showed that the maximal enhancement was obtained at 10 ng/mL (Figure 6B). IL-13 (100 ng/mL) also increased LTA4 hydrolase activity (Figure 7E).
Effect of other cytokines and bioactive substances: IL-6, IL-2, and LPS on LTB4 synthesis in human PMNs To examine the effects of other cytokines and bioactive substances on LTB4 synthesis in human PMNs, we examined the action of IL-6, IL-2, and LPS. LPS significantly inhibited A23187-stimulated production of LTB4 from 2075 ± 125.0 pg/105 cells to 975 ± 110.9 pg/105 cells, and IL-2 tended to decrease it to 1100 ± 108.0 pg/105 cells (P = .08) (Figure 7A). Enzyme protein synthesis, mRNA expression, and enzyme activity for LTA4 hydrolase were also suppressed by LPS treatment (n = 4, P < .05) (Figure 7B-E).Effect of IL-4, IL-10, IL-13, IL-6, IL-2, and LPS on A23187-induced TXB2 production in human PMNs We also examined the action of IL-4, IL-10, IL-13, IL-6, IL-2, and LPS on arachidonate-COX pathway by measuring A23187-stimulated production of TXB2. As shown in Table 2, A23187-stimulated production of TXB2 tended to decline after incubation with IL-4 and IL-13, but the decline was not significant. On the other hand, 6 hours' exposure to LPS (100 ng/mL) enhanced A23187-stimulated production of TXB2 from 605 ± 32.3 pg/106 cells (control) to 917 ± 42.7 pg/106 cells (n = 4, P < .05). Other cytokines showed no significant action on the production of TXB2.
We found that IL-4 and IL-13 increased the production of LTB4 by up-regulating the activity of LTA4 hydrolase in human PMNs. This up-regulation was associated with the increased mRNA expression and new protein synthesis of LTA4 hydrolase.
We would like to thank Professor Daniel Tai for providing anti-LTB4 and anti-TXB2 rabbit sera.
Submitted October 4, 1999; accepted March 6, 2000.
Supported by grants from The Ministry of Education, Science, Sports, and Culture of Japan.
Reprints: Masafumi Zaitsu, Department of Pediatrics, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan; e-mail: g9609{at}post.saga-med.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Abstract
Introduction
