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ková*
*
Department of Pediatrics, Immunology Division, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Canada; and
Cayman Chemical Company, Ann Arbor, MI 48108
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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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Blood eosinophils from asthmatic patients synthesize greater amounts of cysLTs than those from normal subjects (8, 9, 10), and eosinophils appear to be the predominant source of cysLTs in the persistent asthmatic lung (11). CysLTs, in turn, have in vitro chemoattractant activity for human eosinophils (12). Moreover, although the antibronchoconstrictor efficacy of antileukotriene drugs provided the main impetus behind their recent introduction in asthma therapy, clinical trials also provided surprising evidence for a hitherto unsuspected role of cysLTs in promoting persistent eosinophilia in the airways and blood of asthmatics (13, 14). LTD4-induced eosinophil infiltration of the airway could be blocked by the early cysLT antagonist MK-571 (15). More recently, in human studies, blocking the LTD4 receptor has been shown to reduce airway eosinophil numbers in mildly uncontrolled asthma (16), suggesting it may be a contributing factor in lung eosinophilia in asthma.
Eosinophils are known to be important effector cells in IgE-mediated
allergic tissue reactions, including asthma, as well as in immune and
inflammatory reactions against parasitic infections. This capacity is
mediated by the secretion of a variety of immunoregulatory and
inflammatory cytokines, such as GM-CSF, IL-1
, IL-3, IL-5, IL-6,
IL-8, and TNF-, and the chemokine macrophage-inflammatory
protein-1 (reviewed in Ref. 17). Several cytokines,
including IL-3, IL-5, and GM-CSF are reported to regulate the
development of eosinophils from hematopoietic stem cells and to support
their survival in vitro (18, 19, 20, 21). IL-5 is one of the most
important regulators of many eosinophil functions including chemotaxis
(22, 23), degranulation (24, 25), adhesion
(26), and cytotoxicity for parasites (27).
Moreover, treatment of normal eosinophils with IL-5, IL-3, or GM-CSF
enhanced their cysLT production in vitro (28, 29, 30). IL-5 is
also synthesized by Th2 lymphocytes and mast cells in the asthmatic
airway (31, 32, 33) and is detectable in the plasma of
symptomatic asthmatics (34). The importance of IL-5 in
pathogenesis is underscored by the findings that an anti-IL-5 Ab
completely blocks eosinophilic airway infiltration in a mouse model of
asthma (35). Furthermore, mice with targeted disruption of
the IL-5 gene are unable to develop eosinophilic
inflammation of the airways or airway hyperreactivity after allergen
challenge (36). Thus, IL-5 can regulate eosinophilic
inflammation of the airways by modulating multiple functions of
eosinophils.
Recently, Lynch et al. (37) reported the successful cloning and expression of a high-affinity cell surface human LTD4 receptor (CysLT1R) which belongs to the G protein-coupled receptor family. This was soon followed by the publication of Sarau et al. (38), who also reported the cloning and characterization of a CysLTR.
The characterization of leukotriene receptors in human eosinophils, however, is hampered by the limited numbers of eosinophils available from peripheral blood. The promyelocytic leukemia HL-60 cell line can be differentiated toward the eosinophil phenotype (HL-60/eos) when cultured in alkaline medium with sodium butyrate (39, 40). We recently showed that HL-60/eos cells express specific functional LTD4 receptors (41). When they are differentiated toward the eosinophil phenotype, HL-60 cells present several markers and characteristics of mature eosinophils (39, 40). Furthermore, culture in the presence of butyric acid renders HL-60 cells responsive to IL-5, owing to induction of surface IL-5 receptor expression (42). Thus, HL-60/eos cells provide a useful model with which to study the modulation of CysLT1R. In the present study, we set out to determine whether the cytokine IL-5 could modulate the expression of CysLT1R on HL-60/eos cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere with 5% carbon dioxide at 37°C. Differentiation toward the eosinophil phenotype was induced as previously reported (40) by suspending 5 x 105 cells/ml in RPMI 1640 medium containing 10% FBS, antibiotics (as above), 25 mM HEPPS, and 0.5 mM L-butyric acid at pH 7.8. The medium was changed every 3 days. Eosinophil-differentiated HL-60 cells are referred to as HL-60/eos in this text.
RT-PCR
Total RNA was extracted from cells by the guanidine-thiocyanate method described previously (43), and 4 µg of RNA was converted to cDNA by the reverse transcriptase enzyme reaction (AMV transcriptase-reverse; Promega, Madison, WI) in a total volume of 20 µl. PCR was performed in a final volume of 100 µl containing 5 µl of RT reaction product. Samples were placed in a thermal cycler (DNA thermal cycler; Perkin-Elmer, Norwalk, CT) for 34 cycles consisting of 2 min denaturation at 94°C, 30 s annealing at 62°C, and 90 s extension at 72°C, followed by a final 5-min extension at 72°C.
CysLT1R was amplified with the primers derived from the published cDNA sequence for CysLT1R (37), 5'-CGGGATCCGATGAAACAGGAAATC-3' as sense and 5'-CCGGAATTCAATGGGTTTAAACTATAC-3' as antisense. Samples were subjected to parallel amplification of the constitutively expressed, housekeeping gene, GAPDH using the following primers: 5'-GCTAGAGTAAGTAGTT-3' as sense and 5'-AACGACGAGCGTGAC-3' as antisense. A 10-µl aliquot from each PCR was allowed to migrate by electrophoresis in a 1% agarose gel. The CysLT1R-amplified fragment contained 1014 bp. The gel was then colored with ethidium bromide and photographed under UV transillumination. No PCR products were obtained when reverse transcriptase was omitted, indicating that there was no DNA contamination.
Northern blot analysis
In selected experiments, Northern blot analysis was performed to
assess more quantitatively the steady-state levels of
CysLT1R mRNA. Total cellular RNA (20 µg/sample)
was separated by electrophoresis on 1% agarose and transferred onto a
Hybond-N (Amersham, Oakville, Ontario, Canada) membrane for Northern
blot analysis. The cDNA corresponding to the whole coding sequence of
human CysLT1R was amplified by PCR from DNA of
human monocytes. Control hybridizations were performed with the human
GAPDH cDNA probe obtained from the American Type Culture Collection
(Manassas, VA). The probes were labeled with a multiprime DNA-labeling
system (Amersham) using [-32P]dCTP (sp.
act., >3000 Ci/mmol; Amersham). Membranes were prehybridized for
4 h in a mixture containing 120 mM Tris (pH 7.4), 600 mM NaCl, 8
mM EDTA (pH 8.0), 0.1% sodium pyrophosphate, 0.2% SDS, and 100
µg/ml heparin; hybridization was performed overnight at 60°C in the
same mixture in which the concentration of heparin was increased to 625
µg/ml and dextran sulfate at 10% was added. The membranes were then
washed once at room temperature for 20 min in 2x SSC (1x SSC: 0.15 M
NaCl, 0.15 M sodium citrate, pH 7.0); once with 0.1x SSC/0.1% SDS at
60°C for 60 min, and then rinsed at room temperature with 0.1x SSC.
The membranes were exposed to Kodak XAR-5 film (Eastman Kodak,
Rochester, NY) with intensifying screens at -80°C.
Flow cytometry
The expression of CysLT1R in HL-60 and HL-60/eos cells was assessed using a polyclonal anti-CysLT1R Ab directed against the carboxyl-terminal portion of the receptor. The Ab was raised against a peptide corresponding to amino acids 318–337 of the C terminus of human CysLT1R. The peptide was conjugated to keyhole limpet hemocyanin, and rabbits were immunized with 0.2 mg of Ag in CFA. Boosts were done with 0.1 mg of Ag in IFA at 3-wk intervals. Anti-CysLT1R Ab from serum obtained after the fifth boost was purified using a peptide-Sepharose affinity column. The Ab labeled CysLT1R-transfected cells, but not cells transfected with the LTB4 receptor or vector alone. Preincubation of the Ab with the cognate peptide prevented all labeling.
For flow cytometry studies, HL-60 cells were washed with PBS and fixed with 2% paraformaldehyde for 15 min at room temperature followed by permeabilization with 0.1% Triton X-100 for an additional 15 min at room temperature. Cells were resuspended with PBS/2% BSA and labeled for 30 min at 4°C with anti-CysLT1R Ab (or with control, nonpertinent Ab. Cells were then washed with cold PBS and incubated for 30 min at 4°C with FITC-conjugated goat anti-rabbit IgG (BIO/CAN Scientific, Mississauga, Ontario, Canada). Finally, cells were washed again and resuspended in PBS before single-color immunofluorescence analysis of 5000 cells was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). A 1/2000 dilution of the anti-CysLT1R antiserum was used in all cytometry studies.
Intracellular calcium mobilization
For Ca 2+ mobilization assays, 3 x 106 cells were loaded in HBSS (Life Technologies, Rockville, MD), containing 350 mg/l NaHCO3 and 10 mM HEPES (pH 7.0) with the calcium indicator fura-2-acetoxymethyl ester (fura-2-AM; Molecular Probes, Eugene, OR) for 30 min at room temperature. Loaded cells were washed twice, suspended in fresh loading buffer, and added to a constantly stirred cuvette in a SLM/Aminco spectrofluorometer (SLM Instruments, Urbana, IL). The concentration of extracellular Ca2+ was brought to 1.5 mM by addition of a solution of CaCl2 into the cuvette 10 min before recordings. Maximal cell fluorescence (Fmax) was obtained by adding Triton X-100 to a final concentration of 0.5%. Minimal fluorescence (Fmin) was determined by subsequent addition of the chelator EGTA in Tris-HCl buffer (100 mM, pH 9.0) at 125 mM. Stimuli consisted of LTD4, LTB4, and platelet-activating factor (PAF; Cayman Chemicals, Ann Arbor, MI).
Chemotaxis assay
HL-60/eos chemotactic activity was performed with Boyden chambers using a modified Boyden chamber chemotaxis assay. A volume of 200 µl of LTD4 or control medium was added to the lower chamber and 200 µl of HL-60/eos (6 x 10 5) in RPMI 1640 supplemented with 2.5 mg/ml BSA was added to the upper chamber. The two chambers were separated by a 5-µm pore size polycarbonate filter (Osmonics, Westborough, MA). After incubation for 2 h at 37°C in 5% CO2, the filter was disassembled and the upper side of the filter was scraped free of cells. Cells on the lower side were removed with 5 mM EDTA and centrifuged before counting on the FACScan.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-5 induced a
concentration-dependent augmentation of CysLT1R
mRNA expression in HL-60/eos cells. Whereas undifferentiated cells
expressed constitutive CysLT1R mRNA, there was no
modulation with IL-5. Although the amount of
CysLT1R mRNA is generally low in HL-60/eos cells,
Northern blot analysis was nevertheless performed to allow a more
quantitative assessment of CysLT1R mRNA
steady-state expression. As shown in Fig. 2A, IL-5 induced a
time-dependent augmentation of CysLT1R mRNA
expression as early as 2 h of treatment, which persisted at 8
h. Pretreatment of HL-60/eos cells with actinomycin D for 15 min, to
block new RNA synthesis, blocked the IL-5-induced accumulation of
CysLT1R mRNA (Fig. 2B), suggesting a
transcriptional mechanism in the regulation of
CysLT1R expression by IL-5.
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, HL-60/eos readily expressed
CysLT1R protein and the specificity of the
antiserum was demonstrated by blocking of HL-60/eos labeling in the
presence of the cognate peptide. No blocking was seen with an
irrelevant peptide (data not illustrated). IL-5 treatment of HL-60/eos
cells induced a markedly augmented expression of
CysLT1R protein, as compared with untreated cells
(Fig. 4). Although undifferentiated HL-60
cells transcribed CysLT1R mRNA at levels similar
to HL-60/eos cells, they expressed lower levels of
CysLT1R protein at their cell surface and could
not modulate it in response to IL-5.
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A, enhanced
CysLT1R protein expression was detectable by
12 h of treatment, peaked at 24 h, and was still evident at
48 h. Ten nanograms of IL-5 per milliliter induced the strongest
increase in CysLT1R expression (Fig. 5B).
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A, undifferentiated HL-60
cells showed a small response to LTD4, consistent
with their basal expression of CysLT1R, but not
to PAF. HL-60/eos cells, in contrast, readily responded to both
LTD4 and PAF. The response to
LTD4, but not to PAF, was totally blocked by the
selective CysLT1R antagonist MK-571 (Biomol,
Plymouth Meeting, PA). Moreover, the magnitude of the calcium flux was
increased in HL-60/eos cells cultured for 24 h with 10 ng/ml IL-5
in response to LTD4, but not to PAF. As shown in
Fig. 6B, the enhanced responsiveness of IL-5-treated
HL-60/eos cells to LTD4 was associated with both
an increased maximal response
(
[Ca2+]i = 485 ±
109 nM for control cells vs 978 ± 168 nM for IL-5-treated cells,
n = 3) and a reduced EC50 value
(4.1 ± 1.0 nM vs 1.3 ± 0.3 nM) for
LTD4. In counterpart, IL-5 had no significant
effect on the response of HL-60/eos cells to other stimuli, namely,
LTB4 or PAF (Fig. 6C).
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shows the significantly augmented response of IL-5-treated cells to
LTD4. Both an increased number of migrating cells
and a reduced EC50 value (8.8 ± 2.4 nM for
untreated cells vs 0.4 ± 0.1 nM for IL-5-treated cells) for
LTD4 were observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The findings that IL-5 can modulate CysLT1R expression on eosinophilic cells has potential implications in inflammation and asthma. There is substantial evidence linking cytokines with tissue eosinophilia. In allergic inflammatory diseases, the release of several eosinophil-priming cytokines such as IL-5, GM-CSF, and IL-3 is increased (44, 45). An association between the expression of lymphocyte Th2 cytokines, particularly IL-5, and asthma has been demonstrated (31). Elevated levels of IL-5 have been detected in bronchoalveolar lavage fluid obtained 24–48 h following local Ag challenge.
Excessive production of eosinophils and their subsequent invasion of
the airways and other target organs are characteristic features of
asthma and allergic diseases. Although IL-5 seems to have little effect
on the migration of eosinophils into tissues, it has been shown to
prime human eosinophils to respond to other stimuli such as RANTES and
IL-8 (46). Intravenous IL-5 dramatically enhances the
local accumulation of eosinophils induced by intradermal eotaxin or
LTB4 in guinea pigs (47).
Circulating, rather than local, pulmonary IL-5 is required for the
development of Ag-induced airways eosinophilia, possibly through its
action at the level of the bone marrow (48). Since
undifferentiated HL-60 cells are arrested at the promyelocytic stage of
myelopoiesis, it is interesting to observe that
CysLT1R expression is already found in myeloid
precursor cells, although additional studies with primary bone
marrow-derived cells will be necessary to confirm this possibility.
These observations are in contrast to those concerning the receptors
for PAF (49) or LTB4 (our
unpublished observation) which require HL-60 cell differentiation to be
expressed and functional. In fact, eosinophilic differentiation does
not even induce responsiveness to LTB4 (Fig. 6
),
whereas it induces responsiveness to PAF and augments responsiveness to
LTD4. Butyric acid is thought to activate genes
by blocking the actions of deacetylases, thereby inhibiting histone
binding to DNA due to increased histone acetylation (50).
This may lead to the observed induction of IL-5R expression during
eosinophil differentiation with butyric acid (42).
Signaling through IL-5R/IL-5Rß leads to activation of Janus kinase
2 and subsequent phosphorylation and dimerization of STAT factors. IL-5
also activates Lyn, Syk, Raf-1, and PI3 kinases and SHP-2 tyrosine
phosphatase, which are differentially involved in eosinophil growth and
differentiation, survival, up-regulation of adhesion molecules or
priming for degranulation, and production of superoxide and
LTC4 (51). The eventual involvement
of these signaling molecules in the observed up-regulation of
CysLT1R by IL-5 remains to be elucidated.
Anti-inflammatory glucocorticoid therapy reduces IL-5 expression and suppresses airway eosinophilia in asthma (34, 52). Thus, priming of eosinophils with cytokines such as IL-5 and the increase in cysLTs production in the airways of asthmatics may contribute to eosinophil influx and activation.
In summary, the eosinophil-differentiated HL-60 cells can be used as a model for the study of CysLT1R regulation. Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil function and particularly their responsiveness to LTD4, and thus possibly contribute to the pathogenesis of asthma and allergic diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marek Rola-Pleszczynski, Department of Pediatrics, Immunology Division, Faculty of Medicine, Université de Sherbrooke, 3001 North 12th Avenue, Sherbrooke (QC) J1H 5N4, Canada.
3 The abbreviations used in this paper: LT, leukotriene; cysLT, cysteinyl LT; CysLT1R, LTD4 receptor; HL-60/eos, eosinophil-differentiated HL-60 cells; PAF, platelet-activating factor; fura-2-AM, fura-2-acetoxymethyl ester; [Ca2+]i, intracellular calcium concentration.
Received for publication April 27, 2000. Accepted for publication July 31, 2000.
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References |
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in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines. Am. J. Respir. Cell Mol. Biol. 10:471.[Abstract]
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