New induction of leukotriene A₄ hydrolase by interleukin-4 and interleukin-13 in human polymorphonuclear leukocytes

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.

The following reagents were used: RPMI 1640, antibiotic-antimycotic solution (a mixture of penicillin G, streptomycin, and amphotericine B), moloney-murine leukemia virus reverse transcriptase and RNA ladder from Gibco BRL (Grand Island, NY); Ficoll-hypaque, oligo (thymidine [dT]) primer, RNA guard, ethidium bromide, and 100 base-pair (bp) DNA ladder from Pharmacia (Uppsala, Sweden); human type AB serum and diethyl pyrocarbonate (DEPC) from Sigma Chemical (St Louis, MO); Agarose MS-12 from Cosmo Bio (Tokyo, Japan); Norit A (activated charcoal) and EDTA from Wako (Osaka, Japan); recombinant human IL-4, IL-10, and IL-13 from R & D systems Inc (Minneapolis, MN); recombinant human IL-2 and IL-6 from Anapure Bioscientific (Beijing, China);Escherichia coli–derived LPS from Difco Laboratories (Detroit, MI); LTB₄, TXB₂, and 5-HETE from Funakoshi (Tokyo, Japan); [5, 6, 8, 9, 11, 12, 14, 15-³H]–LTB₄ and [5, 6, 8, 9, 11, 12, 14, 15-³H]–TXB₂ from DuPont NEN (Boston, MA); α[³²P] deoxycytidine triphosphate (dCTP) from Toho Chemical (Tokyo, Japan); LTA₄ methylester and antihuman LTA₄ hydrolase rabbit polyclonal antibody from Cayman Chemical (Ann Arbor, MI); antihuman cPLA₂ mouse monoclonal antibody and cPLA₂ standard from Santa Cruz Biotechnology Inc (Santa Cruz, CA); antihuman 5-LO mouse polyclonal antibody from Transduction Laboratories (Lexington, KY); horseradish peroxidase (HRP)–conjugated antirabbit immunoglobulin (Ig)–G goat serum from Jackson Immunoresearch Laboratories Inc (West Grove, PA); HRP-conjugated antimouse IgG goat serum from Kirkegaard and Perry Laboratories Inc (Gaithersburg, MD); deoxyribonucleoside triphosphate (dNTP) mixture and recombinant Taq DNA polymerase from Takara Shuzo (Kyoto, Japan); isogen, random primer labeling kit, and human beta-actin DNA probe from Nippon Gene (Tokyo, Japan); polymerase chain reaction (PCR) primer pairs specific for beta-actin, cPLA₂, 5-LO, 5-lipoxygenase activating protein (FLAP), and LTA₄ hydrolase from Nippon Seifun (Tokyo, Japan); Genescreen from NEN Research Products (Boston, MA). Antisera to LTB₄ and TXB₂ were kindly provided by Dr Tai (University of Kentucky, Lexington, KY). Other materials were all special grade and purchased from commercial sources.

Heparinized venous blood was obtained from healthy volunteers. PMNs were separated as previously described.14 After treating whole blood with 3% dextran sedimentation, we removed erythrocytes by hypotonic lysis. PMNs were obtained by Ficoll-Hypaque gradient centrifugation and washed twice with phosphate-buffered saline (PBS) containing 137 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L NaH₂PO₄, and 1.5 mmol/L K₂PO₄ (pH 7.4). The purity of neutrophil preparation determined by May-Gruward-Giemsa staining was greater than 95%. The viability of cells was checked by trypan blue staining.

The PMNs were incubated in RPMI 1640 containing 5% heat-inactive human type AB serum with various concentrations of IL-4, IL-10, IL-13, IL-6, IL-2, or LPS for the indicated periods at 37°C under 5% CO₂ at a cell density of 2 × 10⁶cells/mL in all studies except for Northern blot, in which cells were incubated at 5 × 106 cells/mL. The cells were recovered after incubation, washed twice with PBS, and used for subsequent studies. In other sets of experiments, cell viability and A23187-stimulated production of LTB₄ were examined after the incubation with IL-4, IL-10, and IL-13 for 0, 6, 9, 15, and 24 hours.

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 100μL 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 TXB₂was 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 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 10⁻⁵ 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 LTB₄diastereoisomers 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.

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).

Complementary DNA (cDNA) was synthesized by reverse transcription-PCR. The reaction mixture (final volume 20 μL) contained 1 μg of total RNA, 1 μg of oligo (dT) primer, 4 μL of 5 × reaction buffer, 2 μL of 0.25 mol/L dithiothreitol, 4 μL of dNTP mixture (each at 2.5 mol/L), 1 μL of RNA guard (5 units), and 1 μL of reverse-transcriptase (200 units).

PCR was carried out in a total volume of 50 μL containing 1 μL of the cDNA, 5 μL of 10 × PCR buffer, 4 μL of dNTP mixture, 0.25 μL of Taq DNA polymerase (5 unit/μL), 100 pmol of paired primers (cPLA₂, 5-LO, FLAP, LTA₄ hydrolase, or beta-actin), and DEPC-treated water. DNA amplification with cPLA₂, 5-LO, FLAP, LTA₄ hydrolase, and beta-actin was performed for 27 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute. The following primers were used: cPLA₂, 5′-ATTCTGGATTGTGCTACCTAC, 5′-AACCAGAAACGCCCAAAACTC; 5-LO, 5′-ACCATTGAGCAGATCGTGGACACGC, 5′-GCAGTCCTGCTCTGTGTAGAATGGG; FLAP, 5′-CACGAAAGCAGGACCCAGAA, 5′-CAGAGCACAGCGAGGAAAGT; LTA₄ hydrolase, 5′-CCACCATCCTTCCCTTAT, 5′-AAACAATCGTCCGCAAAT; beta-actin, 5′-TCCTGTGGCATCACGAAACT, 5′-TAGCAGGTGGCGTTTACGAAG. The size of PCR products were 580 bp in cPLA₂, 488 bp in 5-LO, 116 bp in FLAP, 216 bp in LTA₄ hydrolase, and 314 bp in beta-actin. The PCR products were electrophoresed in 3% agarose gel with the use of TBE buffer (89 mmol/L Tris, 2 mmol/L EDTA, 89 mmol/L sodium borate) and then stained by ethidium bromide. Messenger RNA (mRNA) abundance was visualized under UV exposure and photographed with the use of an image analyzer, Image Master VDS (Pharmacia Biotech; Tokyo, Japan). For every PCR experiment, preliminary experiments were performed to identify the optimum number of cycles of amplification to avoid saturation of the PCR products.

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 LTA₄hydrolase. The DNA fragment was labeled with α[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.

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 × 10⁷cells) 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.

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.

Data were shown as mean ± SEM. Statistical analysis was performed by 1- way analysis of variance. P < .05 was defined as statistically significant.

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 Figure1A, 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 LTB₄production 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).

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 Table1, 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.

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 and3. 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).

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 Figure4A, protein synthesis of LTA₄hydrolase 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 Figure5. IL-13 also increased the amount of LTA₄ hydrolase after 6 hours' incubation at the concentration of 100 ng/mL (Figure 7D).

We further examined the effect of IL-4 on LTA₄hydrolase 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 LTA₄hydrolase activity (Figure 7E).

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) (Figure7A). Enzyme protein synthesis, mRNA expression, and enzyme activity for LTA₄ hydrolase were also suppressed by LPS treatment (n = 4, P < .05) (Figure 7B-E).

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 Table2, 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/10⁶cells (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 LTA₄hydrolase in human PMNs. This up-regulation was associated with the increased mRNA expression and new protein synthesis of LTA₄hydrolase.

IL-4 and IL-13 are produced by Th2 cells, which are dominant in patients with allergic diseases, such as bronchial asthma and atopic dermatitis.1 25-29 IL-4 caused immunoglobulin-class

switching in B-lymphocytes and exerted the immunoglobulin-producing cells to synthesize more IgE.30 IL-4 also induced adhesion molecules on the surface of vascular endothelium and bronchial epithelium and caused migration of eosinophils and neutrophils into the airway in bronchial asthma.4,5,31 Clinical studies demonstrated that the levels of IL-4 were correlated with the numbers of eosinophils in BALF in patients with bronchial asthma, and IL-4 inhalation caused bronchial hyperresponsiveness in these patients.5,29 IL-13 has similar biological functions because the receptor for IL-4 shares many components with that of IL-13.32-35 IL-13 was able to induce immunoglobulin isotype switching to IgE in B-lymphocytes.36 IL-13 also induced adhesion molecules in mouse lung epithelial cells37 and in human lung fibroblasts.31 IL-13 mRNA was up-regulated in bronchial mucosa from patients with bronchial asthma.27Endobronchial allergen challenge increased the concentration of IL-13 in BALF from asthmatic patients.38 Thus, IL-4 and IL-13 play significant roles in the initiation and development of allergic inflammation.

Airway inflammation is an important feature in patients with bronchial asthma.39 The increased number of inflammatory cells was demonstrated in airway secretion and biopsy samples from asthma patients when compared with the control subjects.40-44 The inflammatory cells were not only eosinophils, mast cells, and monocytes but also neutrophils.45-47 Investigators reported that the number of neutrophils in airways was significantly increased in sudden-onset fatal asthma and nocturnal asthma.48-50 Allergen inhalation resulted in acute bronchoconstriction (early allergic response), which was followed by late-phase allergic reaction (LAR) after 6 to 8 hours in sensitized animals and asthma patients. LAR was associated with the accumulation of inflammatory cells, including eosinophils, macrophages, and mast cells.51-53 In the time-course study, neutrophils were increased in the early phase of this event and then replaced by other inflammatory cells.9,47,54 55 These studies suggested that neutrophils might play important roles in developing bronchial hyperreactivity and allergic inflammation by directly injuring airway epithelium and/or by recruiting other inflammatory cells.

The current study has shown that IL-4 and IL-13 significantly enhanced the synthesis of LTB₄ in PMNs. LTB₄ is an arachidonate 5-LO metabolite and a potent chemotactic factor to eosinophils as well as neutrophils. Some investigators reported that the LTB₄ synthesizing activity in peripheral leukocytes was enhanced in bronchial asthma.56-58 We also reported that the level of LTB₄ in BALF obtained from children with severe asthma attacks was significantly higher than that of normal subjects.59 LTB₄ is synthesized predominantly in neutrophils. The synthesis of LTB₄ is initiated by transmembranous stimulation by various factors that increase the level of intracellular calcium ion, leading to the translocation of cPLA₂ from cytosol to nuclear membrane.60,61Cytosolic PLA₂ cleaves AA from membrane phospholipid of the nucleus. The released AA is converted into LTA₄ via an unstable intermediate, 5-hydroperoxyeicosatetraenoic acid, by 5-LO, which is also translocated from cytosol to nuclear membrane where FLAP resides.62,63 The final step is the transformation of LTA₄ to LTB₄ by LTA₄ hydrolase. Thus, LTA₄ hydrolase is a key enzyme of LTB₄synthesis. We previously reported that cPLA₂ is transcriptionally up-regulated by LPS in human PMNs.20 5-LO and FLAP are also newly induced by granulocyte-macrophage colony–stimulating factor in human neutrophils.14,15,64 To our knowledge, there have been no reports that the activity of LTA₄ hydrolase is up-regulated by bioactive substances such as interleukins and growth factors in human neutrophils. In a clinical study, Okano-Mitani et al reported that LTA₄ hydrolase activity was increased in peripheral leukocytes from patients with atopic dermatitis.65 Our demonstration that IL-4 and IL-13 cause a new induction of LTA₄ hydrolase activity reveals a novel up-regulatory mechanism of LTB₄ synthesis in human PMNs. We speculate that IL-4 and IL-13 enhance the transmigration of neutrophils and eosinophils into airway submucosa by increasing the synthesis of LTB₄ and that they amplify the airway inflammation. However, one should be careful to translate the results of the current in vitro study to in vivo phenomena because isolated PMNs are placed in nonphysiologic conditions and A23187 provides no receptor-mediated stimulation.

The enhancement of LTA₄ hydrolase activity in neutrophils is considered to be transcriptionally up-regulated because the human LTA₄ hydrolase gene, which is located in 12q 22, has inducible promoter regions, including phorbol-ester-response element (AP-2) and 2 xenobiotic-response elements.66Further studies are needed to explore more precise mechanisms.

LTA₄ hydrolase is found abundantly in red blood cells as well as neutrophils.67 68 However, red blood cells cannot produce much LTB₄ without transference of LTA₄ from other LTA₄-producing cells because red blood cells have no 5-LO activity. Therefore, neutrophils are the largest source of LTB₄ in humans.

Recent studies revealed that LPS, bacterial endotoxin, increased the synthesis of TXA₂ and PGE₂, arachidonate COX products, by transcriptionally up-regulating COX-2 in human PMNs.20-22 The new induction of COX-2 by LPS was inhibited by exogenously added IL-4 and IL-10.21 Th2 cytokines thus negatively regulate the arachidonate COX pathway and positively regulate the 5-LO pathway in PMNs. Thromboxanes and prostaglandins are the final products of alternative pathway of AA metabolism. AA is alternatively converted by COX-1 and/or COX-2 to PGH₂, which is converted to PGE₂, PGF_2a, PGD₂, PGI₂, and/or TXA₂ depending on cell types. TXA₂ and PGE₂ are the major products in the COX pathway and are proinflammatory lipid mediators produced in PMNs. These biological actions are associated predominantly with the process of nonallergic inflammation caused by bacterial invasion, ischemia, and autoimmune mechanisms. In general, arachidonate COX metabolites are associated with nonallergic inflammation. On the other hand, 5-LO metabolites such as LTC₄, LTD₄, LTE₄, and LTB₄ are predominantly involved in allergic inflammation. It is quite interesting that allergy-associated cytokines IL-4 and IL-13 tend to suppress COX-2 activity and enhance LTA₄hydrolase activity, resulting in the increased production of LTB₄ in Th2 cell–dominant circumstances.

Our study is the first demonstration that Th2-associated cytokines IL-4 and IL-13 up-regulate LTB₄ synthesis in PMNs by increasing LTA₄ hydrolase activity at both the mRNA and the protein levels. Our data suggested that (1) Th2-cell–associated cytokines, IL-4 and IL-13, increase the level of LTB₄production in PMNs in the process of allergic inflammation and (2) PMN-derived LTB₄ may be a key factor amplifying the inflammation. Our results also indicate that the up-regulation of LTB₄ synthesis by IL-4 and IL-13 is specifically attributable to LTA₄ hydrolase, but not to cPLA₂, 5-LO, or FLAP.

We would like to thank Professor Daniel Tai for providing anti-LTB₄ and anti-TXB₂ rabbit sera.