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Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109; and
Division of Pulmonary and Critical Care Medicine, Northwestern University Medical School and Medical Service, Veterans Affairs Chicago Health Care System-Lakeside Division, Chicago, IL 60611
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The synthesis of LTs from arachidonic acid (AA) is initiated by the enzyme 5-lipoxygenase (5-LO), typically working in concert with the 5-LO-activating protein (FLAP). Activation of soluble 5-LO, characterized by its translocation to membranes in a calcium-dependent process (3, 4, 5), positions 5-LO close to its substrate and activating protein. After its activation, 5-LO converts AA to LTA4 in a two-step process. LTA4 is then converted to LTB4 by the enzyme LTA4 hydrolase or to LTC4 by conjugation with glutathione via LTC4 synthase. The generation of LTA4 by 5-LO is a rate-limiting step in LT synthesis. In some cases, changes in LT production have been correlated with changes in the mass of either 5-LO or FLAP (6, 7, 8). However, there are also instances in which enhanced LT secretion has been observed in the absence of changes in the amount of either 5-LO or FLAP (9, 10, 11).
Recent studies have shown that 5-LO can be found in the 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) and alveolar macrophages (16, 17). Furthermore, the compartmentation of 5-LO is not static, as the enzyme rapidly moves from the cytosol into the nucleus in PMN when they migrate from the blood into sites of inflammation in vivo, as well as when they are adhered to various substrates in vitro (18). Interestingly, PMNs recruited into inflammatory sites secrete more LTB4 upon stimulation than PMNs secrete from peripheral blood (18). Thus, the import of 5-LO from the cytosol into the nucleus correlates with increased LT generation in these cells. Similarly, alveolar macrophages, which have intranuclear 5-LO, produce much more LTB4 than their progenitors, peripheral blood monocytes, which have cytosolic 5-LO (17, 19).
Eosinophils (EOS), the predominant infiltrating inflammatory cells in asthma and other allergic diseases, have the capacity to produce large quantities of LTC4 (reviewed in 20 . In addition, data from clinical studies indicate that EOS are a major source of cysteinyl LTs released into the airways in allergic asthma or rhinitis following allergen challenge (21, 22). However, the compartmentation of 5-LO has not been comprehensively examined in this cell type. Because a similar phenomenon in PMNs could be relevant to the overproduction of cysteinyl LTs by EOS, we examined the effect of adherence on the subcellular localization of 5-LO in human EOS in the current study. We then asked whether adherence up-regulates the synthesis of cysteinyl LTs in EOS. We report that although adherence of EOS to fibronectin causes nuclear import of 5-LO, it actually decreases, rather than increases, cysteinyl LT synthetic capacity. The decrease in LTC4 production cannot be ascribed to changes in AA availability, LTC4 synthase activity or oxidative metabolism of LTC4. Instead, the compartmentation of 5-LO within the nucleus appears to protect it from activation, because intranuclear 5-LO translocates poorly to the nuclear envelope upon cell stimulation. As translocation is thought to be essential for 5-LO action, the failure to translocate may explain the diminished metabolism of AA to LTs. These results, taken from in vitro experiments, support a role for nuclear import of 5-LO in regulating cysteinyl LT synthetic capacity in vivo both in normal immune responses and disease states such as asthma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blood for isolation of human EOS was obtained from volunteers
with a clinical history of mild atopic disease (in most cases allergic
rhinitis) and a peripheral eosinophilia of 
3%. These subjects were
all nonsmokers and were not taking oral or inhaled steroids,
nonsteroidal anti-inflammatory drugs, LT synthesis inhibitors, or
LT receptor antagonists. EOS were isolated by a modification of the
method of Hansel et al. (23). Briefly, heparinized venous blood was
centrifuged through Percoll (Pharmacia & Upjohn, Kalamazoo, MI),
(density 1.084 g/ml), and erythrocytes were removed by hypotonic lysis.
The remaining granulocytic fraction was washed, counted, and incubated
with MACS CD16 MicroBeads (Miltenyi Biotec, Auburn, CA) on ice for 30
min. The granulocyte suspension was then applied to type CS magnetic
columns (Miltenyi Biotec) in a 0.6 Tesla magnetic field, and the eluate
containing EOS was collected. Eluates were washed and resuspended in
HBSS containing 2% FCS. The resulting cell suspensions derived by this
method were 97 ± 1% (mean ± SEM; n = 9)
pure EOS and were 98% viable by trypan blue staining.
Cell culture, adherence, and stimulation
For the culture of EOS, individual wells in tissue culture plates or Lab-Tek Permonox chamber slides (Fisher Scientific, Pittsburgh, PA) were coated with human plasma fibronectin (Collaborative Biomedical Products, Bedford, MA) by incubation overnight at 4°C with fibronectin at 100 µg/ml. Then the wells were washed twice with PBS. To block unoccupied binding sites, the wells were incubated for an additional 2 h at 4°C with 0.1% BSA, and washed twice more with PBS. Freshly purified EOS (105 cells in 200 µl) were either maintained in suspension in siliconized tubes or adhered to fibronectin-precoated slides or 96-well plates and incubated at 37°C in 5% CO2/95% air for times ranging from 5 to 120 min. Cells were stimulated by the addition of 2 µM calcium ionophore A23187 (Sigma, St. Louis, MO), or 1 µM FMLP (Sigma) plus 10 µg/ml cytochalasin B (Sigma). In selected experiments, EOS were stimulated with A23187 in the presence or absence of 2 µM diphenylene iodonium (DPI; Cayman Chemical, Ann Arbor, MI). After 10 min, supernatants were removed, microcentrifuged to remove detached cells, and stored at -85°C for subsequent analysis of product formation.
Leukocyte elicitation
Glycogen-elicited rat peritoneal leukocytes were obtained as previously described (18) using respiratory disease-free male Sprague Dawley retired breeder rats (Charles River Laboratories, Portage, MI). Briefly, 30 ml of 1.0% glycogen (Sigma) in saline were introduced into the peritoneum of diethyl ether-anesthetized rats; elicited leukocytes were recovered by peritoneal lavage 4 h after glycogen instillation. Differential counts of cell populations indicated that 3–6% of the cells were EOS.
Indirect immunofluorescent microscopy
As described previously (13), cells that were either maintained in suspension or adhered to fibronectin-coated slides were fixed in -20°C methanol, permeabilized in -20°C acetone, and air dried. Cells were rehydrated and blocked with 1% BSA in PBS containing nonimmune goat serum. Rabbit polyclonal Ab raised against purified human leukocyte 5-LO (a generous gift from Dr. J. Evans, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada; 24 was prepared in 1% BSA-PBS (titer, 1:200) and applied for 1 h, 37°C. Mounts were washed with 1% BSA-PBS and incubated with rhodamine-conjugated goat anti-rabbit Ab (1:200; Sigma) for 1 h, 37°C, then washed extensively and coverslipped. Fluorescence was visualized with a Zeiss Aristoplan microscope equipped for epifluorescence, or imaged by confocal microscopy using a Bio-Rad MRC-600 laser confocal microscope (25). During confocal microscopy, the rhodamine signal was imaged using a 560 nm long pass filter followed by a 585-nm bandpass filter to minimize the contribution due to autofluorescence.
Enzyme immunoassay (EIA) of eicosanoids
Immunoreactive eicosanoids (LTC4, PGE2, thromboxane B2) in conditioned media were quantitated by EIA (Cayman Chemical), according to the supplier’s instructions. Means of data for duplicate wells for each condition were counted as single data points in analyses of multiple experiments.
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 with 0.5 µCi [3H]AA per 5 x 105 cells ml-1 in serum-free RPMI 1640 for 90 min at 37°C. Cells were then washed, resuspended in HBSS + 2% FCS, and plated in fibronectin-precoated 24-well plates at 5 x 105 cells/ml/well. After incubation to allow adherence for 5–120 min, EOS were stimulated by the addition of 2 µM A23187. After 10 min, cultures were acidified to pH 3.0 with 1 N HCl, and media plus cells were extracted with chloroform:methanol as described previously (26). Free [3H]AA was separated by thin layer chromatography (26) and quantitated by liquid scintillation spectrometry. Data are expressed as a percent of the total [3H]AA radioactivity incorporated by EOS, which was determined from parallel unstimulated wells in each experiment.
Whole cell LTC4 synthase assay
EOS (105 cells in 200 µl) were either maintained
in suspension in siliconized tubes or adhered to fibronectin-precoated
96-well plates in HBSS + 2% FCS and incubated at 37°C in 5%
CO2/95% air for 5 or 120 min. LTC4 synthase
was assayed in intact cells by a slight modification of the 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 Chemical) with NaOH under argon according to the
supplier’s instructions immediately before use at each time point. The
reaction was started by addition of LTA4 at a final
concentration of 40 µM and allowed to proceed at 37°C. After 4 min,
media were removed from adherent cultures and immediately
microcentrifuged along with suspension cultures. Supernatants were
removed and stored at -85°C until assay for LTC4 by EIA.
Stimulation and analysis of superoxide production
Freshly purified EOS were adhered to fibronectin-precoated 96-well plates for 5 or 120 min, then stimulated with either 1 µM PMA (Sigma) or 2 µM A23187 in the presence or absence of 2 µM DPI. Superoxide was determined by monitoring the superoxide dismutase inhibitable reduction of ferricytochrome C (Sigma) at 37°C in a THERMOmax microplate reader (Molecular Devices, Menlo Park, CA), as described (28). Duplicate wells were assayed for each condition and the results were averaged.
Evaluation of translocation of nuclear 5-LO
Cells stained for 5-LO were visualized by confocal microscopy,
captured on disk using CoMos software (Bio-Rad Life Sciences,
Hercules, CA), and 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 Image software: the central area within
lobes of nuclei of individual cells, 
625 pixels2 was
densitometrically quantitated. Using the value of 150 or less (on a
scale of 0–255) as a conservative estimation of a darkened field,
nuclei with mean nuclear gray scale values of <150 were defined as
demonstrating translocation of 5-LO from the interior of the nucleus to
the nuclear envelope. Cells from 10 separate fields (20 cells per
field) were scored per slide per condition.
Statistical analysis
In most cases, experiments were repeated at least three times, using different human donors. Values were expressed as means ± SEM. Statistical significance was evaluated by Analysis of Variance (ANOVA), using p < 0.05 as indicative of statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have indicated that adherence of EOS to
fibronectin can alter the regulation of AA metabolism in these cells
(29, 30). For this reason, we examined freshly purified human EOS that
were either maintained in suspension in silicon-treated tubes or
adhered to fibronectin-coated slides before treatment. In cells that
were maintained in suspension and then fixed and stained for 5-LO,
fluorescence was greatest throughout the cytosolic compartment and
notably absent from the bi-lobed nucleus (Fig. 1
A). Punctate fluorescence was
also evident in many cells, suggesting the association of 5-LO with
lipid bodies, as has been reported previously (31). A similar pattern
with the nuclei demonstrating a lack of staining, was observed in EOS
that adhered to fibronectin for only 5 min (Fig. 1B).
Preliminary experiments showed that the granules within fixed EOS
displayed significant autofluorescence when excited with blue light
(seen as a green emission signal; Fig. 1C), and this was
used to further examine the subcellular localization of 5-LO. In a
red-blue-green image analysis, areas of overlap of red and green
signals appear as yellow. The overlay of Fig. 1, B and
C (Fig. 1D) indicated broad colocalization of
5-LO with the granules, as most of each cell appeared yellow and
essentially no 5-LO was associated with the nucleus. By this method, it
was not possible to ascertain whether 5-LO was directly associated with
the granules or merely in the cytoplasm that surrounds the granules.
However, these results are consistent with those of Weller and
colleagues (31), who reported diffuse cytoplasmic staining for 5-LO
obtained by immunocytochemical methods in freshly purified human EOS.
Our results further indicated that there was no significant difference
in the subcellular localization of 5-LO before and immediately after
adherence to fibronectin.
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E), as observed in
freshly purified EOS. However, when EOS were adhered to fibronectin for
120 min and then stained, 5-LO was found predominantly within the
nucleus (Fig. 1F). In all cells, the bi-lobed nucleus was
heavily stained and brighter in fluorescence than the bulk of the
cytosol. In contrast, the autofluorescence of the granules illuminated
the cytoplasmic compartment of adherent EOS excited with blue light
(Fig. 1G). The overlayed images separated the specific
signal of 5-LO again, now in the nucleus from the nonspecific
autofluorescent signal: the cytoplasm, which appeared yellow in briefly
adhered EOS (Fig. 1D), now remained largely green
(Fig. 1H). The redistribution of 5-LO into the nucleus was
evident after adherence for 30 min and appeared to be complete by 60
min (data not shown).
To verify that 5-LO was in fact moving into the bi-lobed nucleus of
EOS, optical sections of individual cells stained for 5-LO were
prepared by confocal microscopy (Fig. 2).
Serial sections of suspension-cultured EOS stained for 5-LO showed
bright cytosolic staining with punctate cytosolic inclusions offset by
deep nonstaining pockets corresponding to the nuclear lobes (Fig. 2A–D). On the other hand, serial sections of EOS
that adhered for 120 min to fibronectin showed 5-LO within the nucleus
in all optical slices (Fig. 2E–H), confirming
the intranuclear localization of the enzyme as opposed to association
only with the nuclear envelope.
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A). Approximately 5% of
these cells, a number corresponding to the percent EOS determined by
differential counting, were characterized by the intense
autofluorescence under blue light that is typical of EOS (Fig. 3B). Thus, as with PMNs, recruited EOS contained
intranuclear 5-LO.
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Recent studies have demonstrated that upon activation cytosolic
5-LO translocates to the nuclear envelope and the associated
endoplasmic reticulum (ER) in peritoneal (14) and alveolar (16, 17)
macrophages, PMN (18, 24), and rat basophilic leukemia cells (16, 25).
When EOS were adhered to fibronectin for 5 min and then stimulated with
the chemotactic peptide FMLP, 5-LO was distributed in a perinuclear
pattern, presumably at the nuclear envelope and ER (Fig. 4A). 5-LO was also evident in
cytosolic inclusions that might correspond to lipid bodies as has been
described (31). In response to stimulation with the calcium ionophore
A23187, 5-LO was abundantly evident at the nuclear envelope and also
within the cell body at a site discrete from the nuclear envelope (Fig. 4B). This extranuclear 5-LO was greatest at the center of
the cell body, consistent with localization on a cytoplasmic membrane
system, most likely the ER. The discrete inclusions, evident in resting
and FMLP-stimulated EOS stained for 5-LO, were absent after stimulation
with A23187.
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A). This is similar to the
enhancement of LTC4 synthesis seen in EOS adhered to
fibronectin as compared with BSA, reported previously by Anwar et al.
(30). Surprisingly, we found that continued incubation on fibronectin
resulted in a significant loss in capacity to release LTC4
upon subsequent stimulation: EOS adhered for 120 min released 10%
of that released by cells adhered for only 5 min. In contrast, cells
maintained in suspension for 120 min under the same conditions 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-associated decline in capacity for synthesis of
LTC4 could not be overcome by stimulating EOS with higher
concentrations of A23187 (up to 10 µM; data not shown). Decreased
LTC4 synthesis in adherent EOS was not due to loss of
viability under these culture conditions, because 98% of cells still
excluded trypan blue after adherence for 120 min.
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A). Also, the addition of
exogenous AA (10 µM) could not overcome the loss of LTC4
synthetic capacity observed with 120-min adherence (Fig. 6B). Finally, adherence to fibronectin was not associated
with a decline in capacity for synthesis of thromboxane or
PGE2 over time (data not shown). These results indicate
that the decline in LTC4 production in adherent EOS does
not result from reduced phospholipase-mediated release of AA.
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). Thus, the
diminished LT production could not be due to reduced LTC4
synthase activity.
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) after adherence for 5 and 120 min. As
shown in Table I, stimulation with PMA
resulted in generation of nearly 10-fold less (O2) by EOS
adhered for 120 min as compared with those adhered for 5 min,
suggesting that the potential for oxidative LT metabolism actually
declines with longer adherence times. Table I also shows that, in
contrast to PMA, A23187 is only a weak stimulus of (O2)
production in EOS. Finally, DPI, an inhibitor of NADPH oxidase (34),
did not prevent the decrease in synthesis of immunoreactive
LTC4 from 120 min adherent EOS, despite the fact that it
effectively blocked (O2) production by PMA-stimulated EOS. Taken
together, these data indicate that the decrease in measured
LTC4 in cultures of EOS that were adhered 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.
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In PMN, the movement of 5-LO into the nucleus was accompanied by a
3- to 5-fold increase in the stimulated LTB4 production in
those cells (18); in EOS, the same movement correlated with a 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 our
previous study (18). Translocation of cytoplasmic 5-LO was also
observable following stimulation of EOS with either FMLP or A23187
after adherence to fibronectin for only 5 min (Fig. 4). However, when
EOS were adhered for 120 min and then stimulated, the intranuclear pool
5-LO rarely exhibited translocation to the nuclear envelope. No cells
showed translocation of intranuclear 5-LO in response to stimulation
with FMLP in the presence of cytochalasin B (Fig. 8A). The vast majority of
cells stimulated with the calcium ionophore A23187 also failed to show
translocation of intranuclear 5-LO (Fig. 8B), although some
darkening of the nuclear compartment that characteristically
accompanies translocation was clearly detectable in some cells (Fig. 8C). Quantitatively, only 8.3 ± 3.7% of EOS
stimulated with 2 µM A23187 showed pronounced translocation of
intranuclear 5-LO. Interestingly, patches of 5-LO fluorescence were
evident at the periphery of nuclei in some A23187-stimulated EOS
(arrows in Fig. 8B), suggesting partial translocation within
these cells. Higher doses of agonist did not enhance 5-LO translocation
in 120 min-adherent EOS (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), and we did not pretreat cells with priming
agents like platelet-activating factor, so we cannot comment on 5-LO
compartmentation in lipid bodies before and after adherence of EOS. An important finding of the current study is that nuclear import of 5-LO, which accompanies prolonged adherence, is associated with a diminished LT synthetic capacity in EOS. This result is particularly surprising in light of the fact that intranuclear compartmentation of 5-LO correlated with enhanced LT synthetic capacity in PMNs after recruitment (18), or in alveolar macrophages (as compared with peritoneal macrophages or blood monocytes) (19). The reduced production of LTC4 in EOS after adherence could not be attributed to changes in the activities of phospholipases (measured as AA release), LTC4 synthase (measured as the conversion of LTA4 to LTC4), or the oxidative metabolism of LTC4. These results highlighted a specific defect in the ability of 5-LO to metabolize AA to LTA4 in adherent EOS.
Translocation of 5-LO has long been recognized as a hallmark of 5-LO action (3, 4, 5). The failure of intranuclear 5-LO to translocate is likely to be a major contributing factor to the reduced LT synthetic capacity of adherent EOS. Whereas release of AA is only slightly decreased in EOS after adherence, the failure of 5-LO to translocate to the nuclear envelope would dissociate 5-LO from both substrate release and the 5-LO activating action of FLAP at that site. It is also possible that the action of intranuclear 5-LO is poorly coupled to that of LTC4 synthase, so that the 5-LO product, LTA4, would be inefficiently converted to LTC4. We have not yet investigated this possibility.
Why doesn’t intranuclear 5-LO translocate to the nuclear envelope? Translocation of 5-LO is a calcium-dependent process (35) and may also require direct phosphorylation of the enzyme by protein tyrosine kinases (36, 37). A failure in either of these two processes may be sufficient to block translocation. In particular, precedents exist for the transient changes in calcium within the nucleus that would be needed to drive 5-LO translocation to be either tightly linked to changes in cytosolic calcium (38, 39) or regulated independently (40). Stimulation of EOS with FMLP or ionophore, as performed in this study, may have been sufficient to cause a calcium transient in the cytosol but not in the nucleus, and as a result activated cytosolic, but not intranuclear, 5-LO. Alternatively, the directed translocation of 5-LO may require additional cofactors or accessory proteins that have yet to be identified, and these may be lacking in the nucleus of EOS.
The current study presents results regarding 5-LO compartmentation and
the regulation of LT synthesis that are provocative and perhaps
paradoxical, particularly in the context of asthma and allergy. Many
atopic individuals, and in particular aspirin-sensitive asthmatics,
have an elevated LTC4 synthetic capacity (41, 42, 43), and
airway EOS are considered to be a major source of LTC4 in
allergic responses and asthma (44). On the other hand, adherence to
matrix proteins like fibronectin is necessary for the recruitment of
EOS into the airway, and we report here that this adherence diminishes,
rather than elevates, LTC4 synthetic capacity. This
apparent conflict may be reconciled through several considerations.
First, our results may reflect the response of recruited EOS in
nonallergic conditions. Therefore, a failure to import 5-LO and thus
decrease LTC4 synthesis during cell recruitment may be a
key element of allergic responses and some types of asthma. The nuclear
import of larger proteins, like 5-LO, requires the presence of a
nuclear import sequence on that protein (45, 46), which may be
activated or deactivated by phosphorylation at a neighboring site on
the same protein (47). A failure to activate the nuclear import
sequence on 5-LO, e.g., by blocking phosphorylation-mediated
activation, could prevent its import during adherence and recruitment,
in effect "locking" the EOS in a high LTC4-producing
mode. This finding would predict that elicited EOS in normal
individuals would show intranuclear 5-LO, as we found in elicited rat
EOS (Fig. 3), whereas in conditions characterized by overproduction of
LTC4, recruited EOS would show cytoplasmic 5-LO. This
possibility is currently under investigation.
A second consideration regarding our findings is that both nuclear import of 5-LO and diminished LT synthesis may be temporary consequences of adherence. That is, with cell adherence for extended periods of time (i.e., greater than 120 min), 5-LO may return to the cytoplasm, resulting in a recovery of LTC4 synthetic capacity. Augmented production of LTC4 may then be driven by extracellular factors, such as platelet-activating factor (31), granulocyte-macrophage CSF (48), IL-3 and IL-5 (49), or TNF (50). As a third consideration, nuclear import of 5-LO may not necessarily result in down-regulation of LTC4 synthetic capacity in vivo. Although we have shown that EOS recruited into the rat peritoneum show intranuclear 5-LO, we have not determined whether they have a diminished capacity to synthesize LTC4, as do cells adhered to fibronectin in vitro. This too may depend on extracellular factors that may have the capacity to prime EOS for LTC4 production. Finally, our results may only apply to our experimental conditions; we already know that they do not necessarily apply to other cell types (e.g., PMNs), and they may differ with alternate matrices or culture conditions. Clearly, further studies, particularly in individuals with asthma or other allergic conditions, are needed to truly assess the relevance of our findings to those conditions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Thomas G. Brock, 6301 MSRB III, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109-0642. E-mail address:
3 Abbreviations used in this paper: LT, leukotriene; 5-LO, 5-lipoxygenase; AA, arachidonic acid; DPI, diphenylene iodonium; EIA, enzyme immunoassay; EOS, eosinophils; FLAP, 5-lipoxygenase-activating protein; PMN, polymorphonuclear neutrophils.
Received for publication July 1, 1998. Accepted for publication October 23, 1998.
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H. Harizi, M. Juzan, J.-F. Moreau, and N. Gualde Prostaglandins Inhibit 5-Lipoxygenase-Activating Protein Expression and Leukotriene B4 Production from Dendritic Cells Via an IL-10-Dependent Mechanism J. Immunol., January 1, 2003; 170(1): 139 - 146. [Abstract] [Full Text] [PDF]
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S. M. Jones, M. Luo, A. M. Healy, M. Peters-Golden, and T. G. Brock Structural and Functional Criteria Reveal a New Nuclear Import Sequence on the 5-Lipoxygenase Protein J. Biol. Chem., October 4, 2002; 277(41): 38550 - 38556. [Abstract] [Full Text] [PDF]
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 C. Bandeira-Melo, L. J. Woods, M. Phoofolo, and P. F. Weller Intracrine Cysteinyl Leukotriene Receptor-mediated Signaling of Eosinophil Vesicular Transport-mediated Interleukin-4 Secretion J. Exp. Med., September 16, 2002; 196(6): 841 - 850. [Abstract] [Full Text] [PDF]
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C. M. Waters, P. H. S. Sporn, M. Liu, and J. J. Fredberg Cellular biomechanics in the lung Am J Physiol Lung Cell Mol Physiol, September 1, 2002; 283(3): L503 - L509. [Abstract] [Full Text] [PDF]
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A. M. Healy, M. Peters-Golden, J. P. Yao, and T. G. Brock Identification of a Bipartite Nuclear Localization Sequence Necessary for Nuclear Import of 5-Lipoxygenase J. Biol. Chem., October 15, 1999; 274(42): 29812 - 29818. [Abstract] [Full Text] [PDF]
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A. S. Cowburn, S. T. Holgate, and A. P. Sampson IL-5 Increases Expression of 5-Lipoxygenase-Activating Protein and Translocates 5-Lipoxygenase to the Nucleus in Human Blood Eosinophils J. Immunol., July 1, 1999; 163(1): 456 - 465. [Abstract] [Full Text] [PDF]
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C. Bandeira-Melo, M. Phoofolo, and P. F. Weller Extranuclear Lipid Bodies, Elicited by CCR3-mediated Signaling Pathways, Are the Sites of Chemokine-enhanced Leukotriene C4 Production in Eosinophils and Basophils J. Biol. Chem., June 15, 2001; 276(25): 22779 - 22787. [Abstract] [Full Text] [PDF]
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T. G. Brock, E. Maydanski, R. W. McNish, and M. Peters-Golden Co-localization of Leukotriene A4 Hydrolase with 5-Lipoxygenase in Nuclei of Alveolar Macrophages and Rat Basophilic Leukemia Cells but Not Neutrophils J. Biol. Chem., September 7, 2001; 276(37): 35071 - 35077. [Abstract] [Full Text] [PDF]
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