Eosinophils Import of 5-Lipoxygenase in Human Blood Accompanies

Decreased Leukotriene C4 Synthesis Accompanies Adherence-Dependent Nuclear Import of 5-Lipoxygenase in Human Blood Eosinophils This information is current as of September 26, 2021. Thomas G. Brock, James A. Anderson, Francine P. Fries, Marc Peters-Golden and Peter H. S. Sporn J Immunol 1999; 162:1669-1676; ; http://www.jimmunol.org/content/162/3/1669 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Decreased Leukotriene C4 Synthesis Accompanies Adherence-Dependent Nuclear Import of 5-Lipoxygenase in Human Blood Eosinophils1

Thomas G. Brock,2* James A. Anderson,† Francine P. Fries,† Marc Peters-Golden,* and Peter H. S. Sporn†

The enzyme 5-lipoxygenase (5-LO) catalyzes the synthesis of leukotrienes (LTs) from arachidonic acid (AA). Adherence or recruitment of polymorphonuclear neutrophils (PMN) induces nuclear import of 5-LO from the cytosol, which is associated with enhanced LTB4 synthesis upon subsequent cell stimulation. In this study, we asked whether adherence of human eosinophils (EOS) causes a similar redistribution of 5-LO and an increase in LTC4 synthesis. Purified blood EOS examined either in suspension or after adherence to fibronectin for 5 min contained only cytosolic 5-LO. Cell stimulation resulted in activation of 5-LO, as evidenced Downloaded from by its translocation to membranes and LTC4 synthesis. As with PMN, adherence of EOS to fibronectin for 120 min caused nuclear import of 5-LO. Unexpectedly, however, adherence also caused a time-dependent decrease in LTC4 synthesis: EOS adhered for 3 120 min produced 90% less LTC4 than did cells adhered for 5 min. Adherence did not diminish the release of [ H]AA from prelabeled EOS or reduce the synthesis of the prostanoids thromboxane and PGE2. Also, inhibition of LTC4 production caused by adherence could not be overcome by the addition of exogenous AA. Adherence increased, rather than decreased, LTC4 synthase activity. However, the stimulation of adherent EOS failed to induce translocation of 5-LO from the nucleoplasm to the nuclear http://www.jimmunol.org/ envelope. This resistance to activation of the nuclear pool of 5-LO with diminished LT production represents a novel mode of regulation of the enzyme, distinct from the paradigm of up-regulated LT synthesis associated with intranuclear localization of 5-LO observed in PMN and other cell types. The Journal of Immunology, 1999, 162: 1669–1676.

eukotrienes (LTs)3 are lipid mediators with central roles with the 5-LO-activating protein (FLAP). Activation of soluble

in the inflammatory response. For example, LTB4 stimu- 5-LO, characterized by its translocation to membranes in a calci- L lates leukocyte chemotaxis, adhesion, superoxide gener- um-dependent process (3–5), positions 5-LO close to its substrate ation, and lysosomal enzyme release (1). The cysteinyl LTs in- and activating protein. After its activation, 5-LO converts AA to by guest on September 26, 2021 cluding LTC4, LTD4, and LTE4 increase microvascular LTA4 in a two-step process. LTA4 is then converted to LTB4 by permeability and mucus secretion and promote the contraction of the enzyme LTA4 hydrolase or to LTC4 by conjugation with glu- smooth muscle (1). However, the overproduction of either LTB or 4 tathione via LTC4 synthase. The generation of LTA4 by 5-LO is a the cysteinyl LTs can contribute to a variety of pathophysiological rate-limiting step in LT synthesis. In some cases, changes in LT processes including asthma and allergic responses (2). Therefore, production have been correlated with changes in the mass of either an understanding of the regulation of LT synthesis is essential to 5-LO or FLAP (6–8). However, there are also instances in which understanding both normal immune responses and the underlying enhanced LT secretion has been observed in the absence of pathophysiology of a variety of diseases. changes in the amount of either 5-LO or FLAP (9–11). The synthesis of LTs from arachidonic acid (AA) is initiated by Recent studies have shown that 5-LO can be found in the the enzyme 5-lipoxygenase (5-LO), typically working in concert cytosol of some resting cells including polymorphonuclear neutrophils (PMN; Refs. 3, 12, and 13) and peritoneal macrophages (14), but in the nucleus of others including mast cells (13, 15) *Division of Pulmonary and Critical Care Medicine, University of Michigan Medical and alveolar macrophages (16, 17). Furthermore, the compart- Center, Ann Arbor, MI 48109; and †Division of Pulmonary and Critical Care Med- icine, Northwestern University Medical School and Medical Service, Veterans Affairs mentation of 5-LO is not static, as the enzyme rapidly moves Chicago Health Care System-Lakeside Division, Chicago, IL 60611 from the cytosol into the nucleus in PMN when they migrate Received for publication July 1, 1998. Accepted for publication October 23, 1998. from the blood into sites of inflammation in vivo, as well as The costs of publication of this article were defrayed in part by the payment of page when they are adhered to various substrates in vitro (18). In- charges. This article must therefore be hereby marked advertisement in accordance terestingly, PMNs recruited into inflammatory sites secrete with 18 U.S.C. Section 1734 solely to indicate this fact. more LTB upon stimulation than PMNs secrete from periph- 1 This work was supported by the American Lung Association (T.G.B.), the Depart- 4 ment of Veterans Affairs (P.H.S.S.), and the Cornelius Crane Asthma Center of North- eral blood (18). Thus, the import of 5-LO from the cytosol into western University (P.H.S.S.). T.G.B. is a Parker B. Francis Fellow in Pulmonary the nucleus correlates with increased LT generation in these Research, an Edward Livingston Trudeau Scholar of the American Lung Association, and Amgen’s Research Grant Awardee. cells. Similarly, alveolar macrophages, which have intranuclear 5-LO, produce much more LTB than their progenitors, periph- 2 Address correspondence and reprint requests to Thomas G. Brock, 6301 MSRB III, 4 Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Cen- eral blood monocytes, which have cytosolic 5-LO (17, 19). ter, Ann Arbor, MI 48109-0642. E-mail address: [email protected] Eosinophils (EOS), the predominant infiltrating inflammatory 3 Abbreviations used in this paper: LT, leukotriene; 5-LO, 5-lipoxygenase; AA, ara- cells in asthma and other allergic diseases, have the capacity to chidonic acid; DPI, diphenylene iodonium; EIA, enzyme immunoassay; EOS, eosinophils; FLAP, 5-lipoxygenase-activating protein; PMN, polymorphonuclear produce large quantities of LTC4 (reviewed in Ref. 20). In addi- neutrophils. tion, data from clinical studies indicate that EOS are a major

source of cysteinyl LTs released into the airways in allergic asthma Indirect immunofluorescent microscopy or rhinitis following allergen challenge (21, 22). However, the As described previously (13), cells that were either maintained in sus- compartmentation of 5-LO has not been comprehensively exam- pension or adhered to fibronectin-coated slides were fixed in Ϫ20°C ined in this cell type. Because a similar phenomenon in PMNs methanol, permeabilized in Ϫ20°C acetone, and air dried. Cells were could be relevant to the overproduction of cysteinyl LTs by EOS, rehydrated and blocked with 1% BSA in PBS containing nonimmune we examined the effect of adherence on the subcellular localization goat serum. Rabbit polyclonal Ab raised against purified human leukocyte 5-LO (a generous gift from Dr. J. Evans, Merck Frosst Centre for of 5-LO in human EOS in the current study. We then asked Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada; Ref. 24) whether adherence up-regulates the synthesis of cysteinyl LTs in was prepared in 1% BSA-PBS (titer, 1:200) and applied for 1 h, 37°C. EOS. We report that although adherence of EOS to fibronectin Mounts were washed with 1% BSA-PBS and incubated with rhoda- causes nuclear import of 5-LO, it actually decreases, rather than mine-conjugated goat anti-rabbit Ab (1:200; Sigma) for 1 h, 37°C, then washed extensively and coverslipped. Fluorescence was visualized with increases, cysteinyl LT synthetic capacity. The decrease in LTC4 a Zeiss Aristoplan microscope equipped for epifluorescence, or imaged production cannot be ascribed to changes in AA availability, LTC4 by confocal microscopy using a Bio-Rad MRC-600 laser confocal mi- synthase activity or oxidative metabolism of LTC4. Instead, the croscope (25). During confocal microscopy, the rhodamine signal was compartmentation of 5-LO within the nucleus appears to protect it imaged using a 560 nm long pass filter followed by a 585-nm bandpass from activation, because intranuclear 5-LO translocates poorly to filter to minimize the contribution due to autofluorescence. the nuclear envelope upon cell stimulation. As translocation is Enzyme immunoassay (EIA) of eicosanoids thought to be essential for 5-LO action, the failure to translocate Immunoreactive eicosanoids (LTC4, PGE2, thromboxane B2) in condi- may explain the diminished metabolism of AA to LTs. These re- tioned media were quantitated by EIA (Cayman Chemical), according sults, taken from in vitro experiments, support a role for nuclear to the supplier’s instructions. Means of data for duplicate wells for each Downloaded from import of 5-LO in regulating cysteinyl LT synthetic capacity in condition were counted as single data points in analyses of multiple vivo both in normal immune responses and disease states such as experiments. asthma. Prelabeling with [3H]AA and stimulation of [3H]AA release In selected experiments, freshly isolated EOS were prelabeled with [3H]AA (60–100 Ci/mmol; DuPont/NEN, Wilmington, DE) by incubating Materials and Methods 3 5 Ϫ1 with 0.5 ␮Ci [ H]AA per 5 ϫ 10 cells ml in serum-free RPMI 1640 for http://www.jimmunol.org/ Preparation of human peripheral blood EOS 90 min at 37°C. Cells were then washed, resuspended in HBSS ϩ 2% FCS, and plated in fibronectin-precoated 24-well plates at 5 ϫ 105 cells/ml/well. Blood for isolation of human EOS was obtained from volunteers with a After incubation to allow adherence for 5–120 min, EOS were stimulated clinical history of mild atopic disease (in most cases allergic rhinitis) and by the addition of 2 ␮M A23187. After 10 min, cultures were acidified to a peripheral eosinophilia of Ն3%. These subjects were all nonsmokers and pH 3.0 with 1 N HCl, and media plus cells were extracted with chloroform: were not taking oral or inhaled steroids, nonsteroidal anti-inflammatory methanol as described previously (26). Free [3H]AA was separated by thin drugs, LT synthesis inhibitors, or LT receptor antagonists. EOS were iso- layer chromatography (26) and quantitated by liquid scintillation spectrom- lated by a modification of the method of Hansel et al. (23). Briefly, hep- etry. Data are expressed as a percent of the total [3H]AA radioactivity arinized venous blood was centrifuged through Percoll (Pharmacia & Up- incorporated by EOS, which was determined from parallel unstimulated john, Kalamazoo, MI), (density 1.084 g/ml), and erythrocytes were wells in each experiment.

removed by hypotonic lysis. The remaining granulocytic fraction was by guest on September 26, 2021

washed, counted, and incubated with MACS CD16 MicroBeads (Miltenyi Whole cell LTC4 synthase assay Biotec, Auburn, CA) on ice for 30 min. The granulocyte suspension was 5 then applied to type CS magnetic columns (Miltenyi Biotec) in a 0.6 Tesla EOS (10 cells in 200 ␮l) were either maintained in suspension in sili- magnetic field, and the eluate containing EOS was collected. Eluates were conized tubes or adhered to fibronectin-precoated 96-well plates in HBSS ϩ washed and resuspended in HBSS containing 2% FCS. The resulting cell 2% FCS and incubated at 37°C in 5% CO2/95% air for 5 or 120 min. suspensions derived by this method were 97 Ϯ 1% (mean Ϯ SEM; n ϭ 9) LTC4 synthase was assayed in intact cells by a slight modification of the pure EOS and were Ն98% viable by trypan blue staining. method of Ali et al. (27) as follows. Before the reaction, 2.5 mM acivicin (Sigma), an inhibitor of ␥-glutamyl transpeptidase, was added to cells.

LTA4 was obtained by hydrolysis of LTA4 methyl ester (Cayman Chem- Cell culture, adherence, and stimulation ical) with NaOH under argon according to the supplier’s instructions immediately before use at each time point. The reaction was started by ad- For the culture of EOS, individual wells in tissue culture plates or Lab-Tek ␮ Permonox chamber slides (Fisher Scientific, Pittsburgh, PA) were coated dition of LTA4 at a final concentration of 40 M and allowed to proceed with human plasma fibronectin (Collaborative Biomedical Products, Bed- at 37°C. After 4 min, media were removed from adherent cultures and ␮ immediately microcentrifuged along with suspension cultures. Superna- ford, MA) by incubation overnight at 4°C with fibronectin at 100 g/ml. Ϫ Then the wells were washed twice with PBS. To block unoccupied binding tants were removed and stored at 85°C until assay for LTC4 by EIA. sites, the wells were incubated for an additional2hat4°Cwith 0.1% BSA, Stimulation and analysis of superoxide production and washed twice more with PBS. Freshly purified EOS (105 cells in 200 ␮l) were either maintained in suspension in siliconized tubes or adhered to Freshly purified EOS were adhered to fibronectin-precoated 96-well plates fibronectin-precoated slides or 96-well plates and incubated at 37°C in 5% for 5 or 120 min, then stimulated with either 1 ␮M PMA (Sigma) or 2 ␮M ␮ CO2/95% air for times ranging from 5 to 120 min. Cells were stimulated A23187 in the presence or absence of 2 M DPI. Superoxide was deter- by the addition of 2 ␮M calcium ionophore A23187 (Sigma, St. Louis, mined by monitoring the superoxide dismutase inhibitable reduction of MO), or 1 ␮M FMLP (Sigma) plus 10 ␮g/ml cytochalasin B (Sigma). In ferricytochrome C (Sigma) at 37°C in a THERMOmax microplate reader selected experiments, EOS were stimulated with A23187 in the presence or (Molecular Devices, Menlo Park, CA), as described (28). Duplicate wells absence of 2 ␮M diphenylene iodonium (DPI; Cayman Chemical, Ann were assayed for each condition and the results were averaged. Arbor, MI). After 10 min, supernatants were removed, microcentrifuged to Evaluation of translocation of nuclear 5-LO remove detached cells, and stored at Ϫ85°C for subsequent analysis of product formation. Cells stained for 5-LO were visualized by confocal microscopy, captured on disk using CoMos software (Bio-Rad Life Sciences, Hercules, CA), and Leukocyte elicitation black and white images were adjusted to full gray scale range (0–255) that defines black as 0 and white as 255. Image files were analyzed using NIH Glycogen-elicited rat peritoneal leukocytes were obtained as previously Image software: the central area within lobes of nuclei of individual cells, described (18) using respiratory disease-free male Sprague Dawley retired ϳ625 pixels2 was densitometrically quantitated. Using the value of 150 or breeder rats (Charles River Laboratories, Portage, MI). Briefly, 30 ml of less (on a scale of 0–255) as a conservative estimation of a darkened field, 1.0% glycogen (Sigma) in saline were introduced into the peritoneum of nuclei with mean nuclear gray scale values of Ͻ150 were defined as dem- diethyl ether-anesthetized rats; elicited leukocytes were recovered by peri- onstrating translocation of 5-LO from the interior of the nucleus to the toneal lavage 4 h after glycogen instillation. Differential counts of cell nuclear envelope. Cells from 10 separate fields (ϳ20 cells per field) were populations indicated that 3–6% of the cells were EOS. scored per slide per condition. The Journal of Immunology 1671

FIGURE 1. Subcellular localization of 5-LO in human EOS. A, Freshly purified cells maintained in suspension for 5 min, fixed, mounted, and then stained for 5-LO. Bar ϭ 10 ␮M for A and E. B–D, Cells adhered to fibronectin for 5 min before fixation and stained for 5-LO. B, Red signal from green excitation (rhodamine indicating 5-LO). Bar ϭ 10 ␮M for B–D. C, Green signal from blue excitation (autofluorescence). D, Overlay of A and B. The overlap of emission signals in this false color pre- sentation appears yellow. E, Control cells maintained in suspension for 120 min, then fixed, mounted, and stained for 5-LO. F–H,

Cells adhered to ﬁbronectin for 120 min. F, Downloaded from Red signal from green excitation (rhodamine indicating 5-LO). Bar ϭ 10 ␮m for F–H. G, Green signal from blue excitation (autoﬂuo- rescence). H, Overlay of F and H. Results are representative of seven independent experiments. http://www.jimmunol.org/

Statistical analysis staining for 5-LO obtained by immunocytochemical methods in In most cases, experiments were repeated at least three times, using dif- freshly purified human EOS. Our results further indicated that by guest on September 26, 2021 ferent human donors. Values were expressed as means Ϯ SEM. Statistical there was no significant difference in the subcellular localization of significance was evaluated by Analysis of Variance (ANOVA), using p Ͻ 5-LO before and immediately after adherence to fibronectin. 0.05 as indicative of statistical significance. To assess the effect of adherence on 5-LO localization in EOS, Results cells were examined after either suspension culture or prolonged adherence to fibronectin-coated slides. When EOS were main- Subcellular localization of 5-LO in human EOS tained in suspension for 120 min, then fixed and stained for 5-LO, Previous studies have indicated that adherence of EOS to fibronec- the enzyme was found solely in the cytoplasmic compartment (Fig. tin can alter the regulation of AA metabolism in these cells (29, 1E), as observed in freshly purified EOS. However, when EOS 30). For this reason, we examined freshly purified human EOS that were adhered to fibronectin for 120 min and then stained, 5-LO were either maintained in suspension in silicon-treated tubes or was found predominantly within the nucleus (Fig. 1F). In all cells, adhered to fibronectin-coated slides before treatment. In cells that the bi-lobed nucleus was heavily stained and brighter in fluores- were maintained in suspension and then fixed and stained for cence than the bulk of the cytosol. In contrast, the autofluorescence 5-LO, fluorescence was greatest throughout the cytosolic compart- of the granules illuminated the cytoplasmic compartment of ad- ment and notably absent from the bi-lobed nucleus (Fig. 1A). herent EOS excited with blue light (Fig. 1G). The overlayed im- Punctate fluorescence was also evident in many cells, suggesting ages separated the specific signal of 5-LO again, now in the nu- the association of 5-LO with lipid bodies, as has been reported cleus from the nonspecific autofluorescent signal: the cytoplasm, previously (31). A similar pattern with the nuclei demonstrating a which appeared yellow in briefly adhered EOS (Fig. 1D), now lack of staining, was observed in EOS that adhered to fibronectin remained largely green (Fig. 1H). The redistribution of 5-LO into for only 5 min (Fig. 1B). Preliminary experiments showed that the granules within fixed EOS displayed significant autofluorescence the nucleus was evident after adherence for 30 min and appeared when excited with blue light (seen as a green emission signal; Fig. to be complete by 60 min (data not shown). 1C), and this was used to further examine the subcellular local- To verify that 5-LO was in fact moving into the bi-lobed nucleus ization of 5-LO. In a red-blue-green image analysis, areas of over- of EOS, optical sections of individual cells stained for 5-LO were lap of red and green signals appear as yellow. The overlay of Fig. prepared by confocal microscopy (Fig. 2). Serial sections of sus- 1, B and C (Fig. 1D) indicated broad colocalization of 5-LO with pension-cultured EOS stained for 5-LO showed bright cytosolic the granules, as most of each cell appeared yellow and essentially staining with punctate cytosolic inclusions offset by deep nonstain- no 5-LO was associated with the nucleus. By this method, it was ing pockets corresponding to the nuclear lobes (Fig. 2A–D). On the not possible to ascertain whether 5-LO was directly associated other hand, serial sections of EOS that adhered for 120 min to with the granules or merely in the cytoplasm that surrounds the fibronectin showed 5-LO within the nucleus in all optical slices granules. However, these results are consistent with those of (Fig. 2E–H), confirming the intranuclear localization of the en- Weller and colleagues (31), who reported diffuse cytoplasmic zyme as opposed to association only with the nuclear envelope. 1672 NUCLEAR IMPORT OF 5-LO IN EOS

FIGURE 4. Subcellular localization of 5-LO in freshly purified EOS after cell stimulation. Cells were adhered to fibronectin for 5 min, then stimulated with either 1 ␮M FMLP plus 10 ␮g/ml cytochalasin B (A)or2 ␮M A23187 (B) for 10 min at 37°C. After removal of the conditioned media, cells were fixed and stained for 5-LO. Bar ϭ 10 ␮m. Results are representative of three independent experiments for FMLP and eight experiments for A23187. Downloaded from 5-LO activation and AA metabolism in adherent human EOS FIGURE 2. Subcellular localization of 5-LO in EOS by optical section- Recent studies have demonstrated that upon activation cytosolic ing. Suspension-cultured (A–D) and 120 min-adherent (E–H) cells were 5-LO translocates to the nuclear envelope and the associated en- stained for 5-LO and imaged by confocal microscopy. Optical slices were doplasmic reticulum (ER) in peritoneal (14) and alveolar (16, 17) ␮ taken at 2- m intervals; images in uppermost panels are closest to the macrophages, PMN (18, 24), and rat basophilic leukemia cells (16, plane of adherence. Bar ϭ 5 ␮m. 25). When EOS were adhered to fibronectin for 5 min and then http://www.jimmunol.org/ stimulated with the chemotactic peptide FMLP, 5-LO was distrib- uted in a perinuclear pattern, presumably at the nuclear envelope Our previous studies with PMNs indicated that recruitment into and ER (Fig. 4A). 5-LO was also evident in cytosolic inclusions sites of inflammation, like adherence, resulted in the import of that might correspond to lipid bodies as has been described (31). In 5-LO from the cytosol into the nucleus (18). To test whether re- response to stimulation with the calcium ionophore A23187, 5-LO cruitment promoted a similar response in EOS, leukocytes were was abundantly evident at the nuclear envelope and also within the recovered from rat peritoneum 4 h after glycogen instillation. Dif- cell body at a site discrete from the nuclear envelope (Fig. 4B). ferential counting of recruited leukocytes following Wright- This extranuclear 5-LO was greatest at the center of the cell body, by guest on September 26, 2021 Giemsa staining indicated that 3–6% of all leukocytes were EOS. consistent with localization on a cytoplasmic membrane system, When unstained samples were prepared for indirect immunofluo- most likely the ER. The discrete inclusions, evident in resting and rescent microscopy, 5-LO was localized to the nucleus of all cells FMLP-stimulated EOS stained for 5-LO, were absent after stim- (Fig. 3A). Approximately 5% of these cells, a number correspond- ulation with A23187. ing to the percent EOS determined by differential counting, were Human EOS isolated by anti-CD16 negative immunomagnetic characterized by the intense autofluorescence under blue light that selection were not stimulated to produce any detectable immuno- is typical of EOS (Fig. 3B). Thus, as with PMNs, recruited EOS reactive LTC4 in the absence of agonist stimulation (data not contained intranuclear 5-LO. shown). On the other hand, EOS stimulated with calcium iono-

phore A23187 produced large amounts of LTC4. Notably, after adherence to ﬁbronectin for 5 min, EOS released about twice as

much LTC4 as cells maintained in suspension for the same time when stimulated with 2 ␮M A23187 (Fig. 5A). This is similar to

the enhancement of LTC4 synthesis seen in EOS adhered to ﬁ- bronectin as compared with BSA, reported previously by Anwar et al. (30). Surprisingly, we found that continued incubation on ﬁ-

bronectin resulted in a signiﬁcant loss in capacity to release LTC4 upon subsequent stimulation: EOS adhered for 120 min released ϳ10% of that released by cells adhered for only 5 min. In contrast, cells maintained in suspension for 120 min under the same con-

ditions released amounts of LTC4 that were similar to those produced by cells in suspension at 5 min. As for EOS stimulated with A23187 (Fig. 5A), cells adhered for 120 min and then stimulated

with FMLP plus cytochalasin B produced about 90% less LTC4 than cells adhered for only 5 min (Fig. 5B). The adherence-asso-

ciated decline in capacity for synthesis of LTC4 could not be overcome by stimulating EOS with higher concentrations of A23187 FIGURE 3. Subcellular localization of 5-LO in recruited leukocytes, ␮ elicited into rat peritoneum. The ﬁeld includes two EOS (arrows) and three (up to 10 M; data not shown). Decreased LTC4 synthesis in ad- PMN. A, The signal from green excitation (rhodamine indicating 5-LO). B, herent EOS was not due to loss of viability under these culture The signal from blue excitation (autoﬂuorescence). Results are represen- conditions, because 98% of cells still excluded trypan blue after tative of four independent experiments. adherence for 120 min. The Journal of Immunology 1673 Downloaded from

FIGURE 5. Effect of adherence to ﬁbronectin on LTC synthesis by 4 FIGURE 6. Effect of adherence time on AA release and AA utilization. EOS. Freshly isolated EOS were suspended in HBSS ϩ 2% FCS, and 1 ϫ 5 Ϫ1

ϫ http://www.jimmunol.org/ 5 A, AA release. EOS (5 10 ml ) in suspension were prelabeled with 10 cells in 200 ␮l were either adhered to fibronectin-coated plastic wells Ϫ [3H]AA (0.5 ␮Ci ml 1) for 90 min, washed, resuspended in HBSS ϩ 2% (open circles) or maintained in suspension in siliconized tubes (filled cir- FCS, and incubated for 10 min with or without 2 ␮M A23187 either in cles) at 37°C and then stimulated at the indicated times. Conditioned media suspension (open bar) or after adherence (filled circles) to fibronectin- were analyzed for LTC by EIA. A, EOS stimulated with 2 ␮M A23187 for 4 coated plastic for the times indicated. Medium was then extracted and 10 min. Results represent means Ϯ SE of six experiments for adherent cells [3H]AA was separated by thin layer chromatography as outlined in Mate- and four experiments for cells in suspension at each time point. By rials and Methods. Results are means Ϯ SE of three experiments per con- ANOVA comparison with cells that adhered 5 min: *, p Ͻ 0.05; **, p Ͻ dition. B, LTC synthesis with or without exogenous AA. Freshly isolated 0.001. B, Stimulation of EOS with 1 ␮M FMLP plus 10 ␮g/ml cytochalasin 4 EOS were suspended in HBSS ϩ 2% FCS, and 1 ϫ 105 cells in 200 ␮l B at 5 and 120 min after adherence to fibronectin. were adhered to fibronectin-coated plastic wells at 37°C for the times indicated. EOS were then stimulated for 10 min with 2 ␮M A23187 in the by guest on September 26, 2021 absence (filled circles) or presence (open circles) of 10 ␮M AA, after Diminished LTC4 production after adherence could possibly result from decreased activity of phospholipase(s), leading to re- which supernatants were collected and analyzed for LTC4 by EIA. Results duced availability of AA, the substrate for 5-LO. However, adher- represent means of data from duplicate samples in one of three similar ence did not significantly affect the release of [3H]AA from experiments. prelabeled EOS stimulated with A23187 (Fig. 6A). Also, the addition of exogenous AA (10 ␮M) could not overcome the loss of times. Table I also shows that, in contrast to PMA, A23187 is only . LTC4 synthetic capacity observed with 120-min adherence (Fig. a weak stimulus of O2 production in EOS. Finally, DPI, an inhib- 6B). Finally, adherence to fibronectin was not associated with a itor of NADPH oxidase (34), did not prevent the decrease in syn- decline in capacity for synthesis of thromboxane or PGE over thesis of immunoreactive LTC from 120 min adherent EOS, de- 2 4 . time (data not shown). These results indicate that the decline in spite the fact that it effectively blocked O2 production by PMA-

LTC4 production in adherent EOS does not result from reduced stimulated EOS. Taken together, these data indicate that the phospholipase-mediated release of AA.

Decreased LTC4 synthesis could also result from reduced conversion of the 5-LO product LTA4 to LTC4 by LTC4 synthase. However, EOS adhered for 120 min showed an enhanced, rather than diminished, ability to convert exogenous LTA4 to LTC4,as compared with cells adhered for only 5 min (Fig. 7). Thus, the diminished LT production could not be due to reduced LTC4 synthase activity.

Finally, decreased measurable LTC4 could result from oxidative degradation by EOS of newly synthesized LT. This has been shown to occur by a myeloperoxidase-dependent mechanism after stimulation of the respiratory burst in isolated human blood PMN FIGURE 7. Effect of adherence to ﬁbronectin on LTC synthase activ- (32, 33). To assess whether oxidative LTC catabolism might oc- 4 4 ity. Freshly isolated EOS were suspended in HBSS ϩ 2% FCS, and 1 ϫ cur in adherent EOS, we determined their capacity for generation . 105 cells in 200 ␮l were either adhered to ﬁbronectin-coated plastic wells of superoxide anion (O2) after adherence for 5 and 120 min. As or maintained in suspension in siliconized tubes at 37°C for 5 or 120 min. shown in Table I, stimulation with PMA resulted in generation of The activity of LTC4 synthase in intact EOS was assessed by the addition nearly 10-fold less O. by EOS adhered for 120 min as compared ␮ 2 of LTA4 (40 M) in the presence of 2.5 mM acivicin, as outlined in Ma- with those adhered for 5 min, suggesting that the potential for terials and Methods. The reaction product, LTC4, was assayed by EIA. The oxidative LT metabolism actually declines with longer adherence results represent means Ϯ SE of results from the two experiments. 1674 NUCLEAR IMPORT OF 5-LO IN EOS

Ϫ Table I. Effect of NADPH oxidase inhibition on O2 generation and LTC4 synthesis in eosinophils adhered for 5 or 120 min

– ␮ 5 5 O2˙ ( mol/min/10 cells LTC4 (pg/10 cells)

5 min 120 min 5 min 120 min

PMA 4.42 0.50 NDb ND PMA ϩ DPI 0.24 0.00 ND ND

A23187 0.08 0.05 3380 248 A23187 ϩ DPI 0.05 0.08 1940 114

a EOS (105/well) were adhered to ﬁbronectin-coated plastic for 5 or 120 min, then stimulated with PMA (1 ␮M) or A23187 (2 ␮M) in the absence or presence of 2 ␮M – DPI. O2˙ generation and LTC4 released into culture supernatants were determined in separate wells (in duplicate) as the SOD-inhibitable reduction of ferricytochrome C and by EIA, respectively. b ND, not done.

decrease in measured LTC4 in cultures of EOS that were adhered Downloaded from for 120 min and stimulated with A23187 cannot be explained by oxidative LT degradation. This, along with the evidence against defects in AA release or LTC4 synthase activity, suggested that the effect of adherence was at the level of 5-LO action. FIGURE 8. Subcellular localization of 5-LO in EOS adhered to fibronectin-coated plastic for 60 min, then washed and stimulated with either Effect of adherence on the translocation of 5-LO FMLP plus cytochalasin B (A) or A23187 (B). An example of translocation http://www.jimmunol.org/ In PMN, the movement of 5-LO into the nucleus was accompanied in response to A23187 stimulation of adherent EOS is also given (C). Arrows indicate sites of patchy accumulation of 5-LO at the edge of the by a 3- to 5-fold increase in the stimulated LTB4 production in those cells (18); in EOS, the same movement correlated with a nucleus. Results are representative of three independent experiments for FMLP and eight experiments for A23187. 90% decrease in stimulated LTC4 production. The translocation of 5-LO from the nucleoplasm to the nuclear envelope was readily detected following stimulation of recruited PMN with A23187 in ingly, in resting blood PMNs, which like EOS are granulocytes, our previous study (18). Translocation of cytoplasmic 5-LO was 5-LO is cytosolic (16), as we have found to be the case in resting also observable following stimulation of EOS with either FMLP or blood EOS in this study. More recently, we have reported that the A23187 after adherence to fibronectin for only 5 min (Fig. 4). by guest on September 26, 2021 subcellular distribution of 5-LO in resting PMNs is dynamic. Thus, However, when EOS were adhered for 120 min and then stimu- when PMNs are recruited into sites of inflammation or are adhered lated, the intranuclear pool 5-LO rarely exhibited translocation to to surfaces, 5-LO moves from the cytosol to the nucleosol (18). the nuclear envelope. No cells showed translocation of intranuclear We report here that a similar shift in subcellular compartmentation 5-LO in response to stimulation with FMLP in the presence of of 5-LO occurs in unstimulated EOS, either after prolonged ad- cytochalasin B (Fig. 8A). The vast majority of cells stimulated with herence or after recruitment into the inflamed rat peritoneum. Fi- the calcium ionophore A23187 also failed to show translocation of nally, 5-LO has recently been localized to cytosolic lipid bodies as intranuclear 5-LO (Fig. 8B), although some darkening of the nu- well as the cytoplasm of freshly purified blood EOS treated in clear compartment that characteristically accompanies transloca- suspension with platelet-activating factor (31). Our preparative tion was clearly detectable in some cells (Fig. 8C). Quantitatively, techniques were not designed to preserve lipid bodies (although only 8.3 Ϯ 3.7% of EOS stimulated with 2 ␮M A23187 showed see Fig. 4), and we did not pretreat cells with priming agents like pronounced translocation of intranuclear 5-LO. Interestingly, platelet-activating factor, so we cannot comment on 5-LO com- patches of 5-LO fluorescence were evident at the periphery of partmentation in lipid bodies before and after adherence of EOS. nuclei in some A23187-stimulated EOS (arrows in Fig. 8B), sug- An important finding of the current study is that nuclear import gesting partial translocation within these cells. Higher doses of of 5-LO, which accompanies prolonged adherence, is associated agonist did not enhance 5-LO translocation in 120 min-adherent with a diminished LT synthetic capacity in EOS. This result is EOS (data not shown). particularly surprising in light of the fact that intranuclear compartmentation of 5-LO correlated with enhanced LT synthetic ca- Discussion pacity in PMNs after recruitment (18), or in alveolar macrophages EOS are the predominant infiltrating inflammatory cells in asthma (as compared with peritoneal macrophages or blood monocytes) and other allergic diseases, and cysteinyl LTs secreted from EOS (19). The reduced production of LTC4 in EOS after adherence are important mediators of the bronchospasm, mucus secretion, could not be attributed to changes in the activities of phospho- and edema that are associated with these diseases. As 5-LO cata- lipases (measured as AA release), LTC4 synthase (measured as the lyzes a rate-limiting step in LT synthesis, understanding its regu- conversion of LTA4 to LTC4), or the oxidative metabolism of lation in EOS is important to understanding the basis for asthma LTC4. These results highlighted a specific defect in the ability of and allergic responses. Recent findings by our lab and others have 5-LO to metabolize AA to LTA4 in adherent EOS. demonstrated that the subcellular distribution of 5-LO among dif- Translocation of 5-LO has long been recognized as a hallmark ferent cell types is heterogeneous. That is, 5-LO is cytosolic in of 5-LO action (3–5). The failure of intranuclear 5-LO to translo- resting monocytes (17) and peritoneal macrophages (14), whereas cate is likely to be a major contributing factor to the reduced LT it is accumulated within the nucleus of alveolar macrophages (17), synthetic capacity of adherent EOS. Whereas release of AA is only mast cells (15), and rat basophilic leukemia cells (16). Interest- slightly decreased in EOS after adherence, the failure of 5-LO to The Journal of Immunology 1675 translocate to the nuclear envelope would dissociate 5-LO from conditions; we already know that they do not necessarily apply to both substrate release and the 5-LO activating action of FLAP at other cell types (e.g., PMNs), and they may differ with alternate that site. It is also possible that the action of intranuclear 5-LO is matrices or culture conditions. Clearly, further studies, particularly poorly coupled to that of LTC4 synthase, so that the 5-LO product, in individuals with asthma or other allergic conditions, are needed LTA4, would be inefficiently converted to LTC4. We have not yet to truly assess the relevance of our findings to those conditions. investigated this possibility. Why doesn’t intranuclear 5-LO translocate to the nuclear enve- Acknowledgments lope? Translocation of 5-LO is a calcium-dependent process (35) and may also require direct phosphorylation of the enzyme by We thank Dr. Jilly Evans for the generous donation of the 5-LO Ab. protein tyrosine kinases (36, 37). A failure in either of these two processes may be sufficient to block translocation. In particular, References precedents exist for the transient changes in calcium within the 1. Lewis, R. A., K. F. Austen, and R. J. Soberman. 1990. Leukotrienes and other nucleus that would be needed to drive 5-LO translocation to be products of the 5-lipoxygenase pathway: biochemistry and relation to pathobiol- ogy in human disease. N. Engl. J. Med. 323:645. either tightly linked to changes in cytosolic calcium (38, 39) or 2. Drazen, J., and K. Austen. 1987. Leukotrienes and airway responses. Am. Rev. regulated independently (40). Stimulation of EOS with FMLP or Respir. Dis. 136:985. 3. Rouzer, C. A., and S. Kargman. 1988. Translocation of 5-lipoxygenase to the ionophore, as performed in this study, may have been sufficient to membrane in human leukocytes challenged with ionophore A23187. J. Biol. cause a calcium transient in the cytosol but not in the nucleus, and Chem. 263:10980. as a result activated cytosolic, but not intranuclear, 5-LO. Alter- 4. Kargman, S., P. Prasit, and J. Evans. 1991. Translocation of HL-60 cell 5-li-

poxygenase. J. Biol. Chem. 266:23745. Downloaded from natively, the directed translocation of 5-LO may require additional 5. Wong, A., M. Cook, S. Hwang, H. Sarau, J. Foley, and S. Crooke. 1992. Stim- cofactors or accessory proteins that have yet to be identified, and ulation of leukotriene production and membrane translocation of 5-lipoxygenase these may be lacking in the nucleus of EOS. by cross-linking of the IgE receptors in RBL-2H3 cells. Biochemistry 31:4046. 6. Pouliot, M., P. McDonald, L. Khamzina, P. Borgeat, and S. McColl. 1994. Gran- The current study presents results regarding 5-LO compartmen- ulocyte-macrophage colony-stimulating factor enhances 5-lipoxygenase levels in tation and the regulation of LT synthesis that are provocative and human polymorphonuclear leukocytes. J. Immunol. 152:851. perhaps paradoxical, particularly in the context of asthma and al- 7. Bennett, C., M. Chiang, B. Monia, and S. Crooke. 1993. Regulation of 5-lipoxygenase and 5-lipoxygenase-activating protein expression in HL-60 cells. Bio- lergy. Many atopic individuals, and in particular aspirin-sensitive chem. J. 289:33. http://www.jimmunol.org/ 8. Stankova, J., M. Rola-Pleszczynski, and C. Dubois. 1995. Granulocyte-macro- asthmatics, have an elevated LTC4 synthetic capacity (41–43), and phage colony-stimulating factor increases 5-lipoxygenase gene transcription and airway EOS are considered to be a major source of LTC4 in al- protein expression in human neutrophils. Blood 85:3719. lergic responses and asthma (44). On the other hand, adherence to 9. Suzuki, K., T. Yamamoto, A. Sato, T. Murayama, R. Amitani, K. Yamamoto, and matrix proteins like fibronectin is necessary for the recruitment of F. Kuze. 1993. Lipopolysaccharide primes human alveolar macrophages for enhanced release of superoxide anion and leukotriene B4: self-limitations of the EOS into the airway, and we report here that this adherence di- priming response with protein synthesis. Am. J. Respir. Cell. Mol. Biol. 8:500. minishes, rather than elevates, LTC4 synthetic capacity. This ap- 10. Steinhilber, D., O. Radmark, and B. Samuelsson. 1993. Transforming growth parent conflict may be reconciled through several considerations. factor ␤ upregulates 5-lipoxygenase activity during myeloid maturation. Proc. Natl. Acad. Sci. USA 90:5984. First, our results may reflect the response of recruited EOS in 11. Petersen, M., R. Steadman, and J. Williams. 1992. Protein kinase C activation by guest on September 26, 2021 nonallergic conditions. Therefore, a failure to import 5-LO and modulates tumour necrosis factor-␣ priming of human neutrophils for zymosan- thus decrease LTC synthesis during cell recruitment may be a key induced leukotriene B4 release. Immunology 75:275. 4 12. Ochi, K., T. Yoshimoto, S. Yamamoto, K. Taniguchi, and T. Miyamoto. 1983. element of allergic responses and some types of asthma. The nu- Arachidonate 5-lipoxygenase of guinea pig peritoneal polymorphonuclear leuko- clear import of larger proteins, like 5-LO, requires the presence of cytes: activation by adenosine 5Ј-triphosphate. J. Biol. Chem. 258:5754. 13. Brock, T. G., R. Paine, and M. Peters-Golden. 1994. Localization of 5-lipoxy- a nuclear import sequence on that protein (45, 46), which may be genase to the nucleus of unstimulated rat basophilic leukemia cells. J. Biol. Chem. activated or deactivated by phosphorylation at a neighboring site 269:22059. on the same protein (47). A failure to activate the nuclear import 14. Peters-Golden, M., and R. McNish. 1993. Redistribution of 5-lipoxygenase and cytosolic phospholipase A2 to the nuclear fraction upon macrophage activation. sequence on 5-LO, e.g., by blocking phosphorylation-mediated ac- Biochem. Biophys. Res. Commun. 196:147. tivation, could prevent its import during adherence and recruit- 15. Chen, X.-S., T. Naumann, U. Kurre, N. Jenkins, N. Copeland, and C. Funk. 1995. ment, in effect “locking” the EOS in a high LTC -producing mode. cDNA cloning, expression, mutagenesis, intracellular localization, and gene chro- 4 mosomal assignment of mouse 5-lipoxygenase. J. Biol. Chem. 270:17993. This finding would predict that elicited EOS in normal individuals 16. Brock, T. G., R. W. McNish, and M. Peters-Golden. 1995. Translocation and would show intranuclear 5-LO, as we found in elicited rat EOS leukotriene synthetic capacity of nuclear 5-lipoxygenase in rat basophilic leuke- (Fig. 3), whereas in conditions characterized by overproduction of mia cells and alveolar macrophages. J. Biol. Chem. 270:21652. 17. Woods, J., M. Coffey, T. Brock, I. Singer, and M. Peters-Golden. 1995. 5-Lip- LTC4, recruited EOS would show cytoplasmic 5-LO. This possi- oxygenase is located in the euchromatin of the nucleus in resting human alveolar bility is currently under investigation. macrophages and translocates to the nuclear envelope upon cell activation. A second consideration regarding our findings is that both nu- J. Clin. Invest. 95:2035. 18. Brock, T., R. McNish, M. Bailie, and M. Peters-Golden. 1997. Rapid import of clear import of 5-LO and diminished LT synthesis may be tem- cytosolic 5-lipoxygenase into the nucleus of neutrophils after in vivo recruitment porary consequences of adherence. That is, with cell adherence for and in vitro adherence. J. Biol. Chem. 272:8276. 19. Bigby, T. D., and M. J. Holtzman. 1987. Enhanced 5-lipoxygenase activity in extended periods of time (i.e., greater than 120 min), 5-LO may lung macrophages compared to monocytes from normal subjects. J. Immunol. return to the cytoplasm, resulting in a recovery of LTC4 synthetic 138:1546. capacity. Augmented production of LTC may then be driven by 20. Weller, P. 1993. Lipid, peptide and cytokine mediators elaborated by eosinophils. 4 In Immunopharmacology of Eosinophils. H. Smith and R. Cook, eds. Academic extracellular factors, such as platelet-activating factor (31), gran- Press, San Diego, p. 25. ulocyte-macrophage CSF (48), IL-3 and IL-5 (49), or TNF (50). 21. Sedgwick, J. B., W. J. Calhoun, G. J. Gleich, H. Kita, J. S. Abrams, As a third consideration, nuclear import of 5-LO may not neces- L. B. Schwartz, B. Volovitz, M. Ben-Yaakov, and W. W. Busse. 1991. Immediate and late airway response of allergic rhinitis patients to segmental antigen chal- sarily result in down-regulation of LTC4 synthetic capacity in vivo. lenge: characterization of eosinophil and mast cell mediators. Am. Rev. Respir. Although we have shown that EOS recruited into the rat perito- Dis. 144:1274. 22. Kane, G., M. Tollino, M. Pollice, C.-J. Kim, J. Cohn, J. Murray, R. Dworski, neum show intranuclear 5-LO, we have not determined whether J. Sheller, J. Fish, and S. Peters. 1995. Insights into IgE-mediated lung inflam- they have a diminished capacity to synthesize LTC4, as do cells mation derived from a study employing a 5-lipoxygenase inhibitor. Prostaglan- adhered to fibronectin in vitro. This too may depend on extracel- dins 50:1. 23. Hansel, T. T., I. J. De Vries, T. Iff, S. Rihs, M. Wandzilak, S. Betz, K. Blaser, and lular factors that may have the capacity to prime EOS for LTC4 C. Walker. 1991. An improved immunomagnetic procedure for the isolation of production. Finally, our results may only apply to our experimental highly purified human blood eosinophils. J. Immunol. Methods 145:105. 1676 NUCLEAR IMPORT OF 5-LO IN EOS

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