From the Department of Medicine, Harvard Thorndike Laboratories, Charles A. Dana Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
Received for publication, February 14, 2001, and in revised form, March 22, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Eosinophils and basophils, when
activated, become major sources of cysteinyl leukotrienes, eicosanoid
mediators pertinent to allergic inflammation. We show that the C-C
chemokines, eotaxin and RANTES (regulated upon activation normal T cell
expressed and secreted), activate eosinophils and basophils for
enhanced leukotriene C4 (LTC4) generation
by distinct signaling and compartmentalization mechanisms involving the
induced formation of new cytoplasmic lipid body organelles.
Chemokine-induced lipid body formation and enhanced LTC4
release were both mediated by CCR3 receptor G protein-linked downstream
signaling involving activation of phosphoinositide 3-kinase,
extracellular signal-regulated kinases 1 and 2, and p38
mitogen-activated protein kinases. Chemokine-elicited lipid body
numbers correlated with increased calcium ionophore-stimulated LTC4 production; and as demonstrated by intracellular
immunofluorescent localization of newly formed eicosanoid, lipid bodies
were the predominant sites of LTC4 synthesis in both
chemokine-stimulated eosinophils and chemokine-primed and
ionophore-activated eosinophils. Eotaxin and RANTES initiated signaling
via phosphoinositide 3-kinase and mitogen-activated protein kinases
both elicits the formation of lipid body domains and promotes
LTC4 formation at these specific extranuclear sites.
Central to the pathogenesis of allergic diseases are
both the recruitment and subsequent activation of specific leukocytes, including notably eosinophils and basophils, at sites of allergic inflammation (1-3). Eosinophils and basophils are major potential sources of cysteinyl leukotrienes
(LTs)1 (LTC4 and
its extracellular derivatives, LTD4 and LTE4),
products of the 5-lipoxygenase (5-LO) pathway of arachidonic metabolism (4). Cysteinyl LTs, as paracrine mediators, cause bronchoconstriction, mucous hypersecretion, increased microvascular permeability, bronchial hyperresponsiveness, and eosinophil infiltration (5-7) and, as autocrine mediators, prolong eosinophil survival (8). Eosinophils and
basophils contain the single LTC4-synthesizing enzyme,
LTC4 synthase, and eosinophils are the predominant cellular
source of this enzyme among resident and recruited cells in the
bronchial tissues of asthmatics (9, 10). In all cells, the highly
regulated generation of LTs is dependent on activation of specific
phospholipases and LT-synthesizing enzymes and involves small molecules
(e.g. Ca2+) and activation-dependent
redistribution of 5-LO to specific membranous compartments within cells
(11). One major candidate mechanism potentially involved in regulating
LT formation is the translocation of 5-LO to the nuclear envelope (10,
11), but in eosinophils this nuclear translocation has been associated with both increased and decreased LTC4 formation (10,
12).
Although mechanisms that activate specific leukocytes to
generate LTs currently focus on the perinuclear envelope as the site of
regulated LT formation (11), eosinophils and other leukocytes associated with inflammatory reactions in vivo
characteristically contain increased numbers of extranuclear lipid-rich
domains in the form of cytoplasmic lipid bodies (13). These enigmatic
organelles, often overlooked if their defining lipid content is lost
during cell staining, have long been noted to be prominent in many cell types associated with inflammation (14, 15). Although lipid bodies lack
a delimiting membrane, their lipid content overlies a poorly understood
honeycomb membranous matrix (16). Neither the genesis nor function of
these organelles is well defined. Although eicosanoid-forming enzymes
have been localized to lipid bodies, including 5-LO and
LTC4 synthase in eosinophil lipid bodies (16, 17), to date
there is no direct evidence that lipid bodies are sites of eicosanoid synthesis.
Both C-C chemokines, eotaxin and RANTES (regulated upon activation
normal T cell expressed and secreted), signaling via CCR3 receptors
expressed on eosinophils (18) and basophils (19), are active in
recruiting these leukocytes to sites of allergic inflammation (20).
Since other chemoattractants can enhance LT formation (21), we
investigated the intracellular pathways by which these chemokines may
activate leukocytes to enhance their regulated formation of cysteinyl
LTs. Notably, chemokine engagement of CCR3 receptors initiated G
protein-linked downstream signaling involving phosphoinositide 3-kinase
(PI3K) and the mitogen-activated protein (MAP) kinases, extracellular
signal-regulated kinases (ERK) 1/2 and p38, to induce the formation of
new lipid body organelles and to enhance eosinophil LTC4
release. Moreover, these cytokines elicited and promoted
LTC4 formation via PI3K and MAP kinase signaling not at the
perinuclear envelope but rather specifically at these extranuclear
lipid body domains.
Eosinophil and Basophil Purification--
Peripheral blood was
obtained with informed consent from 12 normal donors, and eosinophils
were isolated as described (22). Briefly, after anticoagulated blood
was mixed with 6% dextran-saline (MacGaw, Irvine, CA) to facilitate
erythrocyte sedimentation, the leukocyte-enriched plasma was overlaid
onto Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) and
centrifuged at 250 × g for 20 min.
Granulocyte-enriched cell pellets and mononuclear cell-enriched layers
were collected, washed at 4 °C with calcium- and magnesium-free
Hank's balanced salt solution (HBSS Lipid Body Induction and Treatments--
Eosinophil or basophil
suspensions (106/ml) were incubated (37 °C) with eotaxin
(1-100 ng/ml), RANTES (1-100 ng/ml), IL-8 (500 ng/ml) (R&D Systems),
PAF (1 µM) (Calbiochem, La Jolla, CA), or medium alone
for 1 h in HBSS Lipid Body Staining and Enumeration--
Cytospin slides, while
still moist, were fixed with 2% paraformaldehyde in
HBSS LTC4 Measurements--
After samples were taken for
lipid body enumeration, cell suspensions (106/ml) were
washed in HBSS 5-LO Immunolocalization--
Basophil suspensions
(106/ml) were incubated (37 °C) with eotaxin (100 ng/ml)
for 1 h and then cytocentrifuged (500 rpm, 5 min) onto glass
slides. Cytospin preparations were then fixed in 2% paraformaldehyde
for 5 min. After three 10-min washes with 0.05% saponin (Sigma) and
1% human serum (Pierce) in HBSS, slides were incubated for 30 min with
200 µl of polyclonal antiserum anti-5-LO (1:100 dilution) (Cayman) or
with the isotype-matched rabbit IgG control. After washes with HBSS
containing 0.05% saponin and 1% goat serum, slides were incubated for
30 min with Alexa546-labeled goat anti-rabbit IgG antibody (1:100
dilution) (Molecular Probes), washed with 0.1% saponin, washed with
HBSS, and an aqueous mounting medium (Polysciences Inc., Warrington,
PA) was applied to each slide before coverslip attachment. Slides were
viewed with a 100× objective by both phase-contrast and fluorescence
microscopy using the Eclipse TE300 Nikon (Tokyo, Japan) fluorescence
microscope. Electronic photography was performed by spot cooled color
digital camera (model 1.3.0; Diagnostic Instruments Inc., Sterling
Heights, MI) in conjunction with the image editing program Photoshop
(Adobe Systems Inc., San Jose, CA).
Immunodetection of LTC4 at Its Sites of Production in
Eosinophils--
To immunolocalize LTC4 at its formation
sites within eosinophils, viable eosinophils were embedded in an
agarose matrix enabling their morphology and generated products to be
microscopically localized (22). With modifications of prior techniques
(24, 25), water-soluble 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDAC) (Sigma) was used to cross-link eicosanoid carboxyl groups to
amines in adjacent proteins. Briefly, to prepare the agarose matrix,
2.5% agarose (24 °C gelling point) (Promega, Madison, WI) in
sterile distilled H2O was melted at 70 °C; and while
liquid at 37 °C, 9 volumes of agarose were mixed with 1 volume of
10× concentrated RPMI 1640 medium. One volume of this
medium-supplemented agarose was mixed at 37 °C with 3 volumes of
eosinophils at 15 × 106 cells/ml in RPMI 1640 medium
containing 1% fatty acid free-human albumin and 1 volume of RPMI 1640 medium containing 2% fatty acid free-albumin. As indicated, potential
agonists, eotaxin (100 ng/ml), RANTES (100 ng/ml), or IL-8 (500 ng/ml),
were added in 0.1 volumes to agarose/eosinophil mixtures. Immediately
thereafter, 20-µl samples were gently spread onto microscope slides
and covered with CoverWellTM chambers (Grace Bio-Labs, Bend, OR). Each
slide was overlaid with RPMI 1640 medium containing 1% albumin and an identical concentration of the stimulus present in the
agarose/eosinophil mixture. Slides were incubated (37 °C, humidified
5% CO2) for 1 h. Overlying medium was removed and
replaced with RPMI 1640, 1% albumin medium with or without 0.1 µM A23187, and incubated for 15 min (37 °C; 5%
CO2). Incubations were stopped by removing the chambers and
fixing and permeabilizing the cells with 0.5% EDAC in HBSS for 30 min.
In control experiments, eosinophils were alternatively fixed with
paraformaldehyde (2%, 5 min) with and without 0.1% saponin
permeabilization. After three washes (5 min each) with HBSS, the
Alexa488-labeled rat anti-cysteinyl LT detection mAb (clone 6E7; Sigma)
(AlexaTM488 protein labeling using a kit from Molecular Probes,
Eugene, OR) was added (400 µl of 10 µg/ml) for 1 h. Slides
were washed with HBSS, and an aqueous mounting medium (Polysciences)
was applied to each slide before coverslip attachment. Slides were
viewed by both phase-contrast and fluorescence microscopy, and
photography and image analysis were performed as above. Two hundred
eosinophils were scored, and the percentages of those exhibiting green
staining for intracellular immunoreactive LTC4 were calculated.
As a specificity control for the immunolocalization of
LTC4, Alexa488-labeled rat IgG1 (Sigma) was routinely
included as a nonimmune isotype control for the anti-cysteinyl LT
detection antibody. In addition, four other control conditions were
evaluated: (i) substituting paraformaldehyde fixation with and without
saponin permeabilization for the EDAC cross-linking and fixation step; (ii) using neutrophils rather than eosinophils embedded in the gel-matrix; (iii) pre-treating eosinophils in suspension for 30 min
with the 5-LO activating protein inhibitor MK886 (10 µM)
prior to chemokine stimulation, and (iv) pre-treating eosinophils with PI3K inhibitor, wortmannin (1 µM), for 30 min prior to
chemokine stimulation.
Analysis of Intracellular Distribution of Lipid Bodies in Agarose
Matrix-embedded Eosinophils--
To monitor changes in cell
morphology, eosinophils were embedded in the agarose matrix, as
described above, with or without chemokines. Following incubation of
1 h, cells were fixed with 2% paraformaldehyde (5 min) and
stained with OsO4 as described above. To obtain a measure
of polarized redistribution of lipid bodies, cells were divided
approximately into two halves, one of which contained the nucleus.
Lipid bodies were enumerated in the two halves, and those cells found
to have >75% of their lipid bodies in the nuclear half of the cell
were scored as exhibiting polarized lipid body distribution. In four
experiments, 25-50 cells were analyzed.
Statistical Analysis--
Data were expressed as mean ± S.D. Statistical comparisons were done by analysis of variance followed
by Newman-Keuls Student's test. Differences were considered
significant when p < 0.05. Correlation coefficients
were determined by linear regression with significance (F test) at
p < 0.05.
Eotaxin and RANTES Increase and Redistribute Lipid
Bodies--
Resting eosinophils exhibited a spherical shape and a
normal content of osmiophilic lipid bodies (9.2 ± 1.0 lipid
bodies/cell, mean ± S.D., n = 12) distributed
generally throughout the cytoplasm (Fig.
1A). As fully expected from
prior fluid phase assays (18), eotaxin (Fig. 1B) and RANTES
(Fig. 1C), but not the C-X-C chemokine, IL-8 (Fig.
1D), elicited shape changes in eosinophils, including cytoplasmic veiling, increased polarization of their overall shape and
nuclear location, and prominent cytoplasmic projections (lamellipodia and uropodia). Not anticipated by prior results, eotaxin and RANTES also elicited changes in intracellular lipid bodies within eosinophils. Both chemokines dose-dependently induced new lipid body
formation (Fig. 2A). The
magnitude of lipid body induction with each C-C chemokine (at 100 ng/ml) was comparable to that with 1 µM PAF, a recognized
stimulus for leukocyte lipid body formation (16, 23, 26). In contrast,
IL-8 failed to elicit lipid body formation. In eotaxin- and
RANTES-stimulated eosinophils, lipid bodies were located in cytoplasmic
projections and especially in the perinuclear area (Fig. 1,
B and D). In C-C chemokine polarized cells, many lipid bodies became aligned in groups proximate to the nuclear membrane
and in the margin of the trailing uropod. Eosinophils in which >75%
of lipid bodies were in the perinuclear half of the cell were 56 ± 9% with eotaxin (100 ng/ml) (p < 0.05 versus unstimulated, n = 4) and 59 ± 5% with RANTES (100 ng/ml) (p < 0.05 versus unstimulated, n = 3) in contrast to
only 16 ± 7% with unstimulated eosinophils (mean ± S.D.,
n = 4) and 19 ± 6% with IL-8 (500 ng/ml)
(n = 3). Thus, eotaxin and RANTES induced both the
formation of new lipid bodies and their polarized redistribution within
eosinophils.
The capacity of the two chemokines to elicit new formation of
intracellular lipid body organelles was not restricted to eosinophils. Basophils share with eosinophils several functional features including CCR3 expression and the ability to produce LTC4 (19).
Unstimulated basophils contained 3.1 ± 1.0 lipid bodies/cell
(mean ± S.D., n = 4). Both eotaxin and RANTES,
but not IL-8, stimulated new lipid body formation in basophils with
magnitudes comparable to that with 1 µM PAF (Fig.
3, A and B).
The induction of lipid body formation in eosinophils and basophils by
eotaxin and RANTES was mediated through the G protein-linked CCR3
chemokine receptor. Pre-treatment of eosinophils or basophils with
either pertussis toxin or a blocking anti-CCR3 mAb (but not an isotype
control antibody) significantly inhibited lipid body formation induced
by both C-C chemokines (Table I, Fig.
3B). CCR3-initiated downstream signaling pathways active in
lipid body formation included mobilization of intracellular pools of
Ca2+ and PI3K and ERK 1/2 and p38 MAP kinases.
Eotaxin-induced lipid body formation occurred in a
Ca2+-free medium and was blocked (by 82 ± 1%;
n = 3, p < 0.05) by pre-treatment with
the cell permeable Ca2+ chelator, BAPTA-AM (25 µ 77), but
not by its impermeable analog BAPTA free acid (8 ± 8%).
Wortmannin and LY294002, the PI3K inhibitors, PD98059 and U0126, two
specific inhibitors of the ERK1/2 activating kinase (MAP ERK kinase),
and SB203586, a p38 MAP kinase inhibitor, each inhibited
eotaxin-stimulated lipid body formation (Fig.
4A). Thus, eotaxin stimulated
lipid body formation by CCR3-mediated, Gi protein-linked
and PI3K-, ERK 1/2-, and p38 MAP kinase-mediated signaling.
Both exogenous PAF (16, 23) and PAF formed endogenously in eosinophils
in response to IL-5 or Fc Eotaxin and RANTES Enhance Eosinophil and Basophil LTC4
Production--
Both eotaxin and RANTES very effectively primed
eosinophils for increased LTC4 release in response to a
submaximal 0.1 µM concentration of calcium ionophore
A23187 (Fig. 2B). Pre-stimulation of eosinophils for 1 h with eotaxin or RANTES, but not with IL-8, dose-dependently evoked increases in A23187-induced
LTC4 production. At 100 ng/ml, eotaxin- and
RANTES-pre-stimulated eosinophils released about 2.2- and 3.2-fold as
much LTC4 as did eosinophils challenged with A23187 alone.
The increased quantities of LTC4 generated by eosinophils
primed with increasing concentrations of the C-C chemokines correlated
highly with the increased numbers of elicited lipid bodies
(r = 0.91 (p < 0.05) and
r = 0.93 (p < 0.05), for eotaxin and
RANTES, respectively) (Fig. 2, A and B).
Moreover, inhibition of PI3K with wortmannin and LY294002, ERK1/2 with
PD98059 or U0126, and p38 with SB203580, at concentrations that
inhibited eotaxin-induced lipid body formation (Fig. 4A),
also inhibited LTC4 production by eosinophils (Fig.
4B). Again, inhibitors of protein kinase C or tyrosine
kinase, which did not inhibit eotaxin-induced lipid body formation
(Fig. 4A), also failed to affect the consequent LTC4 production (Fig. 4B).
Although eosinophils stimulated for 1 h with 100 ng/ml eotaxin or
RANTES formed more lipid bodies, quantities of LTC4
released extracellularly in supernatants from eosinophils not activated by the calcium ionophore were not sufficient to be detectable by enzyme
immunoassays (EIA) (data not shown). Supernatants of eotaxin- or
RANTES-stimulated basophils also did not contain sufficient levels of
LTC4 to be detectable by EIA, but both C-C chemokines effectively primed basophils for increased LTC4 release in
response to A23187. At 100 ng/ml, eotaxin- and RANTES-pre-stimulated basophils released 8.1- and 9.3-fold, respectively, as much
LTC4 as did basophils challenged with A23187 alone.
Moreover, pertussis toxin and anti-CCR3 neutralizing antibody
blocked both lipid body induction (Fig. 3B) and enhanced
LTC4 production by basophils stimulated with eotaxin and
RANTES (data not shown).
Lipid Bodies Are Sites for Eotaxin- and RANTES-enhanced
LTC4 Synthesis in Eosinophils--
Since eotaxin- and
RANTES-initiated signaling led to correlative quantitative increases in
both lipid body formation and enhanced LTC4 formation, we
employed a new strategy for direct in situ immunolocalization of intracellular LTC4 to ascertain the
intracellular compartmentalization of cysteinyl LT synthesis.
Unstimulated eosinophils exhibited no immunofluorescent staining for
LTC4 (Table II, Fig. 5A), demonstrating no
background staining or pre-formed LTC4, as expected in the
absence of cell stimulation. In contrast, eosinophils activated for 15 min with 0.1 µM calcium ionophore A23187 yielded intense
and localized immunofluorescent staining for LTC4 in
virtually all eosinophils (Table II, Fig.
6A). Much of the staining was in a linear perinuclear pattern, although sites distant from the nuclei
also exhibited LTC4 labeling. The specificity of this
immunofluorescent staining for LTC4 was ascertained. First,
the detected LTC4 should be a product of an active 5-LO
pathway in eosinophils; accordingly MK886, which blocks 5-LO pathway
activity, completely abolished immunofluorescent staining for
LTC4 in A23187-stimulated eosinophils (Table II, Fig.
6A). Second, specificity of the immunofluorescence for
cysteinyl LTs was supported by the absence of immunostaining when a
fluorochrome-labeled isotype control antibody replaced the
anti-cysteinyl LT mAb (Table II). Third, A23187-stimulated neutrophils
(which do not form cysteinyl LTs) exhibited no immunofluorescent labeling with the fluorochrome-labeled anti-cysteinyl LT mAb (Table II). Fourth, substitution of paraformaldehyde fixation with and without
saponin permeabilization for EDAC treatment abolished immunofluorescent
detection of LTC4 in A23187-stimulated eosinophils, indicating that the cell permeabilization and eicosanoid lipid-protein cross-linking effected by EDAC were essential for intracellular LTC4 immunodetection (data not shown). The detecting
fluorochrome-labeled mAb recognizes LTC4, LTD4,
and LTE4, but the latter two are formed extracellularly,
whereas only LTC4 is generated intracellularly (6). These
findings validate the specificity for detecting LTC4 formed
at sites within stimulated eosinophils.
About 25% of eosinophils stimulated with eotaxin or RANTES exhibited
staining for immunoreactive LTC4 (Fig. 5, B and
C), that was fully inhibitable by the 5-LO inhibitor, MK886,
and the PI3K inhibitor, wortmannin (Table II). In contrast, eosinophils
incubated with IL-8 exhibited no anti-LTC4
immunofluorescence (Table II, Fig. 5D). Although occasional
perinuclear LTC4 localization was observed in
eotaxin-stimulated (Fig. 5B, arrowhead) and
RANTES-stimulated eosinophils, most LTC4 was localized at
punctate locations (Fig. 5, B and C, arrows)
distant from the nucleus. Thus, in the absence of calcium ionophore
stimulation, the two C-C chemokines were directly stimulating low level
LTC4 formation within eosinophils, although extracellular
levels remained beneath the sensitivity of LTC4 EIA assays
of supernatant fluids (as noted above). This chemokine-stimulated
LTC4 formation occurred predominantly at discrete
extranuclear lipid body sites. Moreover, LTC4 formation at
lipid bodies was dependent on PI3K activation.
Further evidence of the roles of lipid bodies as sites of
LTC4 formation was found in eosinophils that were first
primed with eotaxin or RANTES and then stimulated with calcium
ionophore to activate the 5-LO pathway. Whereas unprimed and
non-polarized eosinophils stimulated with A23187 exhibited substantial
linear, perinuclear rim anti-LTC4 staining, consistent with
localization at the nuclear envelope (Fig. 6A,
arrowheads), and some focal staining at extranuclear sites,
the pattern of LTC4 localization was different in
eotaxin- and RANTES-primed eosinophils. In these C-C chemokine-primed
and A23187-stimulated eosinophils, only occasional LTC4
immunofluorescence was detected with a linear perinuclear rim pattern
(Fig. 6C, arrowhead). More extensive
anti-LTC4 staining exhibited a punctate pattern, with very
distinct focal staining proximate to, but separate from, the nucleus
(Fig. 6B, asterisk) and within the uropodia and
lamellipodia (Fig. 6, B and C, arrows)
of polarized eosinophils, fully consistent in size, form, number, and
distribution with cytoplasmic lipid bodies (Fig. 1, B and
C). Again, pre-treatment of eosinophils with the 5-LO pathway inhibitor, MK886, completely abolished all LTC4
immunostaining (Fig. 6, B and C). The PI3K
inhibitor, wortmannin, which inhibited chemokine enhanced release of
LTC4 from eosinophils (Fig. 4), inhibited
chemokine-elicited LTC4 production only at all (both preformed and chemokine-elicited) lipid body sites, with no inhibitory effect on the immunoreactive LTC4 generated at the
perinuclear membrane of eosinophils (Fig. 6). Notably, in
A23187-activated eosinophils, wortmannin inhibited lipid body
LTC4 formation, but inhibited neither LTC4
production at the perinuclear membrane (Fig. 6) nor overall
LTC4 generation, as assessed by immunofluorescent microscopy (Table II) and ELISA assays of released LTC4
(data not shown).
In basophils, LTC4 localization at lipid bodies was not
possible since these cells were destroyed during EDAC cross-linking and
fixation, but basophil lipid bodies were sites of 5-LO localization (Fig. 3C).
Our results elucidate mechanisms whereby chemoattractrant
chemokines may activate a major functional response in eosinophils and
basophils, specifically their capacity to generate cysteinyl LTs, and
also provide novel findings pertinent to the regulated intracellular
compartmentalization of eicosanoid formation by multiple cell types.
The C-C chemokines, eotaxin and RANTES, acting via CCR3 receptors on
eosinophils and basophils, may not only recruit these leukocytes to
sites of allergic inflammation but also activate distinct intracellular
signaling and compartmentalization mechanisms within these recruited
cells to enhance their capacity to form cysteinyl LTs. The means
whereby these chemokines enhance eosinophil and basophil LT formation
were suggested by the finding that these two C-C chemokines, but not
IL-8, stimulated the formation of new cytoplasmic lipid bodies,
organelles previously implicated in eicosanoid synthesis (17). This
induction of lipid body formation was mediated via G protein-linked
CCR3 chemokine receptors and was dependent on downstream activation of
PI3K and the ERK1/2 and p38 MAP kinases. Activation of ERK and p38 MAP
kinases in eosinophils in response to chemoattractants (21) and of PI3K in human basophils in response to IgE-mediated stimulation (29) has
been shown to participate in the regulated activation of
LTC4 formation, but the means for such regulation has been
undefined. Inhibitors of PI3K, ERK1/2, and p38 MAP kinases inhibited
both CCR3-mediated lipid body formation and CCR3-mediated priming for enhanced eosinophil LTC4 release. CCR3-mediated downstream
signaling, in contrast to IL-5- and Fc That the formation of new lipid body organelles elicited by
CCR3-mediated signaling provided a distinct intracellular compartment for the regulated generation of LTC4 was indicated by
several findings. The numbers of eotaxin- and RANTES-elicited lipid
bodies correlated with the magnitudes of the priming responses for
increased extracellular release of LTC4 elicited by prior
exposure of eosinophils to eotaxin and RANTES. Inhibitors of PI3K and
ERK1/2 and p38 MAP kinases inhibited both CCR3-mediated lipid body
formation and enhanced LTC4 release by eosinophils. More
directly, newly formed LTC4 was localized almost
exclusively to lipid bodies in eotaxin- and RANTES-stimulated
eosinophils, in contrast to perinuclear localization of
LTC4 in eosinophils activated solely with calcium ionophore
A23187. In ionophore-activated eosinophils, the PI3K inhibitor,
wortmannin, failed to inhibit the overall eosinophil LTC4
generation that was shown to occur at perinuclear membranes. In
contrast, wortmannin, which inhibited the chemokine-enhanced extracellular release of LTC4, blocked chemokine-stimulated
LTC4 formation at both preformed and chemokine-elicited
lipid bodies, but not at the perinuclear membrane. These findings
indicated that chemokine-elicited activation of PI3K was involved in
both the induction of new lipid body formation and the regulated
activation of LTC4 formation at all lipid body domains,
providing additional evidence that chemokine-elicited lipid bodies were
the principal sites of enhanced LTC4 synthesis.
Heretofore, the intracellular sites of eicosanoid formation in any cell
have not been directly demonstrated, but rather have been inferred
based on the immunolocalization of specific eicosanoid-forming enzymes.
Hence, the translocation of 5-LO from the cytosol to the nucleus in
eosinophils in response to calcium ionophore has suggested that
enhanced LTC4 is formed at perinuclear sites (12) (consistent with our findings in eosinophils solely activated by
ionophore). Indeed, in many cells, major sites of synthesis of both
5-LO- and cyclooxygenase pathway-derived eicosanoids are believed to be
the perinuclear membranes (11, 30, 31). Based on calcium
ionophore-elicited translocation of 5-LO from either the nucleus or the
cytosol, redistribution of 5-LO to the nuclear envelope has been found
in neutrophils, eosinophils, alveolar macrophages, blood monocytes,
mast cells, and the rat basophilic leukemia mast cell-like cell line
(11). A role for extranuclear sites for 5-LO catalyzed LT formation,
however, would be compatible with the earlier finding that enucleate
neutrophil cytoplasts generate LTB4 in response to A23187
(32). More recently, we established that enucleate eosinophil
cytoplasts formed lipid bodies in response to PAF stimulation and that
the numbers of lipid bodies in cytoplasts correlated with levels of
primed LTC4 and PGE2 released by cytoplasts
following submaximal A23187 challenge (16). Moreover, lipid bodies in
enucleate cytoplasts were sites of immunolocalized 5-LO,
cyclooxygenase, and LTC4 synthase proteins (16). Our
findings extend knowledge of the regulated intracellular
compartmentalization of eicosanoid formation. Although perinuclear
membranes may be sites of eicosanoid formation in cells singularly
activated with calcium ionophore A23187 (as we confirmed), in
leukocytes first stimulated with specific chemokines or other leukocyte
agonists, e.g. PAF, lipid bodies (as present in
vivo in inflammation-associated leukocytes) are the predominant sites of enhanced eicosanoid formation.
Since the immunolocalization of eicosanoid-forming proteins need not
reflect the regulated activity of these enzymes, we utilized a more
direct approach to detect the intracellular sites of 5-LO- and
LTC4 synthase-mediated LTC4 formation in
eosinophils. Eosinophils incubated in a gel matrix were fixed and
permeabilized with a water soluble cross-linker EDAC, enabling: 1) the
covalent cross-linking of eicosanoid carboxyl groups to adjacent amines
in proteins at the sites of eicosanoid formation, 2) the penetration of
detecting fluorochrome-conjugated anti-cysteinyl LT mAb into
eosinophils, and, importantly, 3) the relative preservation of lipid
body domains (which dissipate with air drying or commonly used alcohol
fixation). Eosinophils were especially amenable to this strategy for
localizing LTC4, their dominant 5-LO product. In
eosinophils, 5-LO and LTC4 synthase enzymatic activities
are tightly coupled, as indicated by the paucity of nonenzymatic
breakdown products of intermediate LTA4 (33); both enzymes
have been localized at eosinophil lipid bodies (16). In basophils, 5-LO
was also localized at lipid bodies; but basophils, unlike eosinophils,
were not durable to the LTC4 fixation process. In contrast,
in calcium ionophore-activated neutrophils, 5-LO-catalyzed formation of
LTA4 is not tightly coupled to subsequent LTB4
formation, which may be mediated by cytosolic leukotriene
A4 hydrolase or may occur substantially following extracellular release of LTA4 (34). Immunofluorescent
localization of newly formed LTC4 in eosinophils activated
with calcium ionophore exhibited a predominantly perinuclear staining,
remarkably similar to the immunolocalization of 5-LO protein reported
in ionophore-activated eosinophils (12). In contrast, eosinophils
exposed to eotaxin or RANTES prior to ionophore activation showed
predominant anti-LTC4 staining at focal lipid body
structures either proximate to the nucleus or in distant lamellipod
protrusions. Moreover, wortmannin inhibition of PI3K blocked both
overall eotaxin-primed eosinophil LTC4 release and lipid
body (but not perinuclear) LTC4 formation. Thus, in C-C
chemokine-primed and ionophore-activated eosinophils, lipid body
organelles were the predominant sites of regulated LTC4 synthesis.
Lipid bodies are complex and as yet poorly understood organelles. They
are not unique to eosinophils and basophils and are found in a
diversity of cells ranging from fibroblasts to endothelial cells to
leukocytes (35). Lipid body numbers characteristically increase in
cells associated with various forms of inflammation (14, 15, 36). Lipid
body formation is rapidly inducible by specific stimuli, including
hypoxia in endothelial cells (37), cis-unsaturated fatty
acids in neutrophils (38), and receptor-mediated ligands, including PAF
(23) and, as shown here, specific chemokines for eosinophils and
basophils. Lipid bodies contain esterified arachidonic acid, ill
defined membranous structures, and in many cell types, including
eosinophils, basophils, neutrophils, alveolar macrophages, mast cells,
one or more enzymes involved in eicosanoid synthesis, including
cyclooxygenase, 5-LO, 15-LO, and/or LTC4 synthase (16,
39-41). Lipid bodies also contain cytosolic phospholipase A2 and several signal transducing kinases, including PI3K,
ERK1, ERK2, p85, and p38 MAP kinases (42, 43). Although these findings have suggested that lipid bodies might be sites of regulated eicosanoid formation in several cell types, direct evidence for such had been
lacking. Our direct demonstration that eosinophil lipid bodies are
specific sites of LTC4 synthesis has implications for many other cells types, especially those involved in diverse inflammatory responses, in which lipid bodies might likewise serve as sites of
regulated eicosanoid formation.
Although eosinophils stimulated with eotaxin or RANTES did not release
levels of LTC4 adequate for detection in supernatants by
EIA, the heightened sensitivity of the immunofluorescent detection of
LTC4 formation enabled the demonstration that about a
quarter of eosinophils stimulated solely with these C-C chemokines, and not with IL-8, synthesized low levels of LTC4. Since
LTC4 is being recognized to have autocrine effects on
eosinophils, including prolonging their longevity (8) and regulating
the vesicular transport-mediated release of preformed eosinophil
granule-derived cytokines (e.g.
IL-4),2 the capacity of the
two chemokines to stimulate even low level LTC4 synthesis
intracellularly at focal extranuclear sites may augment eosinophil
effector functioning. Moreover, the stimulation of eosinophils for
increased LTC4 formation by eotaxin and RANTES, based on
their receptor-mediated signaling to induce and activate lipid body
organelles that are sites for regulated LTC4 synthesis, promotes cysteinyl LT generation and release by recruited eosinophils and further contributes to the pathogenesis of allergic inflammation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
), and depleted of
erythrocytes by hypotonic saline lysis. Eosinophils and basophils were
negatively selected using the MACS system (Miltenyi Biotec, Auburn, CA)
with anti-CD16 plus anti-CD3 or a mixture of immunomagnetic beads,
respectively. The viability of freshly isolated cells was >95% (by
trypan blue exclusion), eosinophil purity was >99% (by
HEMA3® staining, Fisher Scientific, Pittsburgh, PA), and
basophil purity was 75-85%. Purified cell suspensions were adjusted
to 1 × 106 or 15 × 106 cells/ml in
HBSS
/
containing 0.1% endotoxin-free ovalbumin or RPMI
1640 medium containing 1% fatty acid free-human albumin (Sigma)
for use in fluid- or gel-phase assays, respectively.
/
and then cytocentrifuged (500 rpm, 5 min) onto glass slides. For inhibitor studies, cells were
pre-treated for 30 min with anti-CCR3 mAb (clone 61828.111; R&D
Systems) or isotype control rat IgG2a at 10 µg/ml (PharMingen, San
Diego, CA), pertussis toxin (100 ng/ml) (Calbiochem), the PAF receptor
antagonist CV6209 (10 µM), the 5-LO inhibitor AA861 (10 µM), the 5-LO activating protein inhibitor MK886 (10 µM), the PKC inhibitors chelerythrine (10 µM) and calphostin C (1 µM), the PI3K
inhibitors wortmannin (1 µM) and LY294002 (10 µM), the tyrosine kinase inhibitors herbimycin (10 µM) and genistein (10 µM), or the MAP
kinase inhibitors PD98059, U0126, and SB203580 (each 10 µM) (Biomol, Plymouth, PA), or their vehicles, as
indicated. Stock solutions of stimuli and inhibitors were prepared in
HBSS
/
containing 0.1% endotoxin-free ovalbumin,
aliquoted, and stored at
20 °C. AA861, MK886, calphostin C,
herbimycin, genistein, PD98059, U0126, SB203580, and A23187 were
diluted in Me2SO. The final Me2SO concentration
was <0.01% and had no effect on eosinophils.
/
, rinsed in 0.1 M cacodylate buffer
(pH 7.4), stained in 1.5% OsO4 (30 min), rinsed in
distilled H2O, immersed in 1% thiocarbohydrazide (5 min),
rinsed with 0.1 M cacodylate buffer, restained with 1.5% OsO4 (3 min), and then dried and mounted (23). Lipid bodies were enumerated by light microscopy with a 100× objective lens in 50 consecutively scanned cells.
/
, resuspended in 1 ml of HBSS containing
calcium and magnesium, and then stimulated with 0.1 µM
A23187 (Sigma) for 15 min (37 °C). Reactions were stopped on ice,
cell suspensions were centrifuged (500 × g for 10 min;
4 °C), and supernatants were assayed for LTC4 by enzyme
immunoassay (EIA) (sensitivity < 7.8 pg/ml) (Cayman Chemical, Ann
Arbor, MI).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (118K):
[in a new window]
Fig. 1.
Intracellular distribution of lipid bodies in
chemokine-stimulated eosinophils. Eosinophils were incubated for
1 h with medium alone (A), eotaxin (100 ng/ml)
(B), RANTES (100 ng/ml) (C), or IL-8 (500 ng/ml)
(D). Lipid bodies were stained with osmium, and eosinophils
were visualized by both phase-contrast (left
panels) and light microscopy (right
panels). Arrows indicate representative lipid
bodies localized in lamellipodia, and arrowheads indicate
lipid bodies closely localized around the nucleus in eotaxin- and
RANTES-stimulated eosinophils. Bar, 5 µm. Images are
representative of five independent experiments.
View larger version (42K):
[in a new window]
Fig. 2.
Lipid body formation (A) and
priming for LTC4 production (B) by C-C
chemokine-stimulated human eosinophils. Eosinophils were incubated
with indicated chemokines or PAF for 1 h for induction of lipid
bodies. Thereafter, eosinophils were incubated with 0.1 µM A23187 for 15 min for LTC4 production. To
normalize for differences in base-line LTC4 release between
different donors, results are presented as percentage of increase in
the LTC4 released by A23187-stimulated cells. Results are
means ± S.D. from three to five independent assays. *,
p < 0.05; **, p < 0.01, compared with
negative controls.
View larger version (90K):
[in a new window]
Fig. 3.
C-C chemokines induce formation of lipid
bodies containing 5-LO in human basophils. Basophils were
incubated for 1 h with medium alone, eotaxin (100 ng/ml),
RANTES (100 ng/ml), or IL-8 (500 ng/ml). In A, images of
representative basophils stained with osmium and visualized by both
phase-contrast (left panels) and light microscopy
(right panels) are shown. In B, lipid
bodies were enumerated in 50 consecutive cells, and each value
represents the mean ± S.D. of lipid bodies from one experiment
representative of three experiments. C shows
immunolocalization of 5-LO at lipid bodies in eotaxin-stimulated
basophils. Focal, punctate staining of lipid bodies is present with
5-LO antiserum (upper panel) and absent with
non-immune control (lower panel)
dashed lines denote base-line levels with medium
alone. PTX, pertussis toxin.
C-C chemokine-induced lipid body formation in eosinophils is mediated
via CCR3 receptor signaling, but not by endogenous PAF or 5-LO
pathway derivatives
View larger version (40K):
[in a new window]
Fig. 4.
Signaling via activation of PI3K and ERK1/2
and p38 MAP kinases mediates lipid body formation (A)
and priming for LTC4 production (B) by C-C
chemokine-stimulated human eosinophils. Eosinophils were
pre-treated for 30 min with kinase inhibitors (as indicated) and then
incubated with eotaxin for 1 h for induction of lipid bodies.
Thereafter, eosinophils were incubated with 0.1 µM A23187
for 15 min for LTC4 production. To normalize for
differences in base-line LTC4 release between different
donors, results are presented as percentage of increase in the
LTC4 released by A23187-stimulated cells. Results are
means ± S.D. from four independent assays. **, p < 0.01 compared with eotaxin.
receptor engagement (26) also signal via
pertussis toxin-inhibitable G protein-linked PAF receptors to elicit
lipid body formation. For PAF-induced lipid body formation, post-PAF
receptor downstream signaling is obligately dependent on 5-LO
activation to form 5-(S)-hydroxyeicosatetraenoic acid (23).
We, therefore, evaluated whether CCR3 downstream signaling to form
lipid bodies was mediated either by autocrine PAF formation or
dependent on endogenous 5-LO activation. Concentrations of the PAF
receptor antagonist CV6209, the 5-LO inhibitor AA861, and the 5-LO
activating protein inhibitor MK886 that blocked PAF-induced lipid body
formation in eosinophils did not inhibit lipid body formation induced
by the C-C chemokines (Table I). Other downstream signaling for lipid
body formation also differed for PAF- and CCR3-mediated responses.
Although PAF-elicited lipid body formation is inhibitable by the
protein kinase C inhibitors, chelerythrine and calphostin C (27, 28),
these inhibitors did not block CCR3-mediated lipid body formation in
eosinophils (Fig. 4A). The tyrosine kinase inhibitors,
herbimycin and genistein, inhibited neither CCR3-elicited (Fig.
4A) nor PAF-elicited (27) lipid body formation. Thus, the
intracellular signaling pathways leading to lipid body induction differ
between the PAF and CCR3 chemokine receptors, although both are G
protein-linked, seven-transmembrane-spanning receptors that are
expressed on eosinophils and basophils and can initiate lipid body formation.
Specificity and sensitivity of detection of immunoreactive LTC4
in eosinophils
View larger version (58K):
[in a new window]
Fig. 5.
C-C chemokines directly stimulate
LTC4 production in eosinophils. Phase-contrast
(left panels) and fluorescent (right
panels) microscopy of identical fields of eosinophils
incubated for 1 h, and then fixed with EDAC and stained with
Alexa488-labeled anti-cysteinyl LT mAb. To facilitate intracellular
localization, anti-LTC4 immunoreactive sites
(green staining) were overlaid on phase-contrast
images and white lines were drawn delineating the
nuclear perimeter. Unstimulated (A) and IL-8-stimulated
(D) eosinophils display no fluorescent LTC4
immunostaining. Representative eosinophils stimulated with eotaxin (100 ng/ml) (B) and RANTES (100 ng/ml) (C) exhibit
perinuclear (arrowheads) and punctate cytoplasmic
(arrows) immunoreactive LTC4. Bar, 5 µm.
View larger version (68K):
[in a new window]
Fig. 6.
Perinuclear and extranuclear production of
LTC4 in human eosinophils. Phase-contrast and
fluorescent microscopy of identical fields of eosinophils incubated for
1 h with the C-C chemokines (as indicated) and activated for 15 min with 0.1 µM A23187. Eosinophils were fixed with EDAC
and stained with Alexa488-labeled anti-cysteinyl LT mAb. To facilitate
intracellular localization, anti-LTC4 immunoreactive sites
(green staining) were overlaid on phase-contrast
images and white lines were drawn delineating the
nuclear perimeter. Eosinophils activated with A23187 exhibit
fluorescent anti-LTC4 staining especially at the
perinuclear envelope (arrowheads) as well as at some
extranuclear sites (arrow). Eosinophils stimulated with
eotaxin (100 ng/ml) (B) or RANTES (100 ng/ml) (C)
prior to A23187 activation exhibited some perinuclear
anti-LTC4 staining (arrowheads) and abundant
punctate LTC4 immunoreactive staining both proximate to the
nucleus (*) and at more distant cytoplasmic sites (arrows).
Eosinophils pre-treated with the 5-LO inhibitor, MK 886 (10 µM), had no anti-LTC4 immunofluorescent
staining (right panels), whereas cells
pre-treated with the PI3K inhibitor, wortmannin, showed only
perinuclear staining for LTC4. Bar, 5 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor-mediated stimulation
of eosinophil lipid body formation (26), did not require endogenous PAF
formation and, distinct from PAF, which signals through its own
heterotrimeric G-protein-linked, seven-transmembrane-spanning receptor,
was not dependent on intermediate 5-LO activity or PKC activation
(23).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Bruno L. Diaz and Anne Nicholson-Weller for comments on the work and manuscript and Lesley Woods for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AI20241, AI22571, AI41995 and HL56386.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Beth Israel
Deaconess Medical Center, DA-617, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-3307; Fax: 617-277-6061; E-mail:
pweller@caregroup.harvard.edu.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M101436200
2 C. Bandeira-Melo, M. Phoofolo, and P. F. Weller, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LT, leukotriene; PI3K, phosphoinositide-3 kinase; 5-LO, 5-lipoxygenase; MAP, mitogen-activated protein; ERK, extracellullar signal-regulated kinase; EIA, enzyme immunoassay; HBSS, Hank's balanced salt solution; PAF, platelet activating factor; RANTES, regulated upon activation normal T cell expressed and secreted; IL, interleukin; EDAC, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide; mAb, monoclonal antibody.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Wardlaw, A. J. (1999) J. Allergy Clin. Immunol. 104, 917-926[Medline] [Order article via Infotrieve] |
2. | Gleich, G. J. (2000) J. Allergy Clin. Immunol. 105, 651-663[Medline] [Order article via Infotrieve] |
3. | Costa, J. J., Weller, P. F., and Galli, S. J. (1997) J. Am. Med. Assoc. 278, 1815-1822[Abstract] |
4. | Weller, P. F. (1993) in Immunopharmacology of Eosinophils: The Handbook of Immunopharmacology (Smith, J. H. , and Cook, R. M., eds) , pp. 25-42, Academic Press, London |
5. | Lewis, R. A., Austen, K. F., and Soberman, R. J. (1990) N. Engl. J. Med. 323, 645-655[Medline] [Order article via Infotrieve] |
6. |
Henderson, W. R., Jr.
(1994)
Ann. Intern. Med.
121,
684-697 |
7. | Laitinen, L. A., Haahtela, T., Spur, B. W., Laitinen, A., Vilkka, V., and Lee, T. H. (1993) Lancet 341, 989-990[Medline] [Order article via Infotrieve] |
8. |
Lee, E.,
Robertson, T.,
Smith, J.,
and Kilfeather, S.
(2000)
Am. J. Respir. Crit. Care Med.
161,
1881-1886 |
9. |
Cowburn, A. S.,
Sladek, K.,
Soja, J.,
Adamek, L.,
Nizankowska, E.,
Szczeklik, A.,
Lam, B. K.,
Penrose, J. F.,
Austen, F. K.,
Holgate, S. T.,
and Sampson, A. P.
(1998)
J. Clin. Invest.
101,
834-846 |
10. |
Cowburn, A. S.,
Holgate, S. T.,
and Sampson, A. P.
(1999)
J. Immunol.
163,
456-465 |
11. |
Peters-Golden, M.,
and Brock, T. G.
(2000)
Am. J. Respir. Crit. Care Med.
161,
S36-S40 |
12. |
Brock, T. G.,
Anderson, J. A.,
Fries, F. P.,
Peters-Golden, M.,
and Sporn, P. H.
(1999)
J. Immunol.
162,
1669-1676 |
13. | Weller, P. F., and Dvorak, A. M. (2000) in Asthma and Rhinitis (Busse, W. W. , and Holgate, S. T., eds) , pp. 351-372, Blackwell Scientific Publications, Boston |
14. | Robinson, J. M., Karnovsky, M. L., and Karnovsky, M. J. (1982) J. Cell Biol. 95, 933-842[Abstract] |
15. | Coimbra, A., and Lopes-Vaz, A. (1971) J. Histochem. Cytochem. 19, 551-557[Medline] [Order article via Infotrieve] |
16. |
Bozza, P. T., Yu, W.,
Penrose, J. F.,
Morgan, E. S.,
Dvorak, A. M.,
and Weller, P. F.
(1997)
J. Exp. Med.
186,
909-920 |
17. | Weller, P. F., Bozza, P. T., Yu, W., and Dvorak, A. M. (1999) Int. Arch. Allergy Immunol. 118, 450-452[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Sabroe, I.,
Hartnell, A.,
Jopling, L. A.,
Bel, S.,
Ponath, P. D.,
Pease, J. E.,
Collins, P. D.,
and Williams, T. J.
(1999)
J. Immunol.
162,
2946-2955 |
19. |
Uguccioni, M.,
Mackay, C. R.,
Ochensberger, B.,
Loetscher, P,
Rhis, S.,
LaRossa, G. J.,
Rao, P.,
Ponath, P. D.,
Baggiolini, M.,
and Dahinden, C. A.
(1997)
J. Clin. Invest.
100,
1137-1143 |
20. |
Rothenberg, M. E.
(1999)
Am. J. Respir. Cell Mol. Biol.
21,
291-295 |
21. |
Bates, M. E.,
Green, V. L.,
and Bertics, P. J.
(2000)
J. Biol. Chem.
275,
10968-10975 |
22. | Bandeira-Melo, C., Gillard, G., Ghiran, I., and Weller, P. F. (2000) J. Immunol. Methods 244, 105-115[CrossRef][Medline] [Order article via Infotrieve] |
23. | Bozza, P. T., Payne, J. L., Goulet, J. L., and Weller, P. F. (1996) J. Exp. Med. 183, 1515-1525[Abstract] |
24. | Ogawa, H., Kassell, N. G., Sasaki, T., Hongo, K., Tsukahara, T., Hudson, S. B., Asban, G. I., Tuan, H. L., and Torner, J. C. (1988) Prostaglandins 36, 891-900[Medline] [Order article via Infotrieve] |
25. | Liu, L. X., Buhlmann, J. E., and Weller, P. F. (1992) Am. J. Trop. Med. Hyg. 46, 520-523[Medline] [Order article via Infotrieve] |
26. |
Bartemes, K. R.,
McKinney, S.,
Gleich, G. J.,
and Kita, H.
(1999)
J. Immunol.
162,
2982-2989 |
27. | Bozza, P. T., Yu, W., Cassara, J., and Weller, P. F. (1998) J. Leukocyte Biol. 64, 563-569[Abstract] |
28. |
Bozza, P. T.,
Payne, J. L.,
Morham, S. G.,
Langenbach, R.,
Smithies, O.,
and Weller, P. F.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
11091-11096 |
29. |
Miura, K.,
and MacGlashan, D. W., Jr.
(2000)
Blood
96,
2199-2205 |
30. | Smith, W. L., DeWitt, D. L., and Garavito, R. M. (2000) Annu. Rev. Biochem. 69, 145-182[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Spencer, A. G.,
Woods, J. W.,
Arakawa, T.,
Singer, I. I.,
and Smith, W. L.
(1998)
J. Biol. Chem.
273,
9886-9893 |
32. | Haines, K. A., Giedd, K. N., and Weissmann, G. (1986) Biochem. J. 233, 583-588[Medline] [Order article via Infotrieve] |
33. | Weller, P. F., Lee, C. W., Foster, D. W., Corey, E. J., Austen, K. F., and Lewis, R. A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7626-7630[Abstract] |
34. |
Sala, A.,
Bolla, M.,
Zarini, S.,
Muller-Peddinghaus, R.,
and Folco, G.
(1996)
J. Biol. Chem.
271,
17944-17948 |
35. | Galli, S. J., Dvorak, A. M., Peters, S. P., Schulman, E. S., MacGlashan, D. W., Jr., Isomura, T., Pyne, K., Harvey, V. S., Hammel, I., Lichtenstein, L. M., and Dvorak, H. F. (1985) in Prostaglandins, Leukotrienes, and Lipoxins (Bailey, J. M., ed) , pp. 221-239, Plenum Publishing Co., New York |
36. | Triggiani, M., Oriente, A., Seeds, M. C., Bass, D. A., Marone, G., and Chilton, F. H. (1995) J. Exp. Med. 182, 1181-1190[Abstract] |
37. |
Scarfo, L. M.,
Weller, P. F.,
and Farber, H. W.
(2001)
Am. J. Physiol.
280,
H294-H301 |
38. | Weller, P. F., Ryeom, S. W., Picard, S. T., Ackerman, S. J., and Dvorak, A. M. (1991) J. Cell Biol. 113, 137-146[Abstract] |
39. | Dvorak, A. M., Morgan, E., Tzizik, D. M., and Weller, P. F. (1994) Int. Arch. Allergy Immunol. 105, 245-250[Medline] [Order article via Infotrieve] |
40. | Dvorak, A. M., Schleimer, R. P., Dvorak, A. M., Lichtenstein, L. M., and Weller, P. F. (1992) Int. Arch. Allergy Immunol. 99, 208-217 |
41. | Weller, P. F., Monahan-Earley, R. A., Dvorak, H. F., and Dvorak, A. M. (1991) Am. J. Pathol. 138, 141-148[Abstract] |
42. | Yu, W., Bozza, P. T., Tzizik, D. M., Gray, J. P., Cassara, J., Dvorak, A. M., and Weller, P. F. (1998) Am. J. Pathol. 152, 759-769[Abstract] |
43. |
Yu, W.,
Cassara, J.,
and Weller, P. F.
(2000)
Blood
95,
1078-1085 |