| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI
mRNA expression, or
their ability to sustain basophil viability in culture. We also examine
a variety of the signaling steps that accompany activation with these
cytokines. In contrast with the ability of IL-3 to alter
secretagogue-mediated cytosolic calcium responses following 18-h
cultures, 18-h treatment with IL-5 or NGF did not affect C5a-induced
leukotriene C4 generation or alter C5a-induced intracellular
Ca2+ concentration elevations. IL-3 and IL-5, but
not NGF, induced FcRI mRNA expression and all three improved
basophil viability in culture with a ranking of IL-3 > IL-5
NGF. All three cytokines acutely activated the extracellular
signal-regulated kinase pathway and the signaling elements that
preceded extracellular signal-regulated kinase and cytosolic
phospholipase A2 phosphorylation, consistent with their
redundant ability to acutely prime basophils. However, only IL-3 and
IL-5 induced Janus kinase 2 and STAT5 phosphorylation. This pattern of
signal element activation among the three cytokines most closely
matched their ability to induce expression of FcRI mRNA.
Induction of the sustained calcium signaling that follows overnight
priming with IL-3 appeared to be related to the strength of the early
signals activated by these cytokines but the relevant pathway required
was not identified. None of the signaling patterns matched the ability
of the cytokines to promote basophil survival. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
For studies of human basophils, we have used as a model of the priming
effect induced by IL-3 the ability of IL-3 to enhance LTC4 release
following stimulation with C5a. C5a is a strong secretagogue for
histamine release but for basophils from most donors does not induce
LTC4 release (15, 16, 17). Preincubation of the cells with
cytokines (IL-3, IL-5, GM-CSF, and NGF), for only 5–15 min,
"permits" C5a to induce marked LTC4 release (3, 4, 16, 17). Although each of these cytokines appears to prime the
basophil—with respect to LTC4 release—to a similar extent following a
short incubation, it is also known that receptor expression for IL-3,
IL-5, and GM-CSF, i.e., those receptors with 
subunits that share a
common subunit, is markedly different for these three cytokines.
This raises the possibility that even between these three cytokines
that not all priming effects are equivalent. With respect to NGF, which
does not share the same subunit construction as IL-3, IL-5, and GM-CSF,
it also seemed likely that priming might be qualitatively distinct
beyond the initial acute effects. We hypothesized that this array of
cytokines is not strictly redundant in their actions, and that it would
be possible to distinguish among IL-3, IL-5, and NGF on the basis of
the other changes induced in basophils. There are already indications
that this is true: IL-3, but not IL-5, is capable of inducing IL-13
secretion. IL-3 synergistically potentiates C5a-induced secretion of
IL-13 from human basophils whereas IL-5 is far less effective
(18). IL-3, but not IL-5, potentiates Ag-induced, or a
combination of Ag- and eotaxin-induced, IL-4 production from these
cells (19). In an effort to better understand the
significance of the multiple cytokines present during allergic
inflammation, the current studies further define some of the
distinguishing characteristics of three cytokines, IL-3, IL-5, and NGF,
and explore some of the relevant signaling steps that distinguish the
activities of these three cytokines.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following were purchased: PIPES, BSA, EGTA, EDTA,
D-glucose, NaF,
Na4P2O7,
Na3VO4, 2-ME, Nonidet P-40,
Tris-HCl (Sigma, St. Louis, MO); crystallized human serum albumin (HSA)
(Miles Laboratories, Elkhart, IN); FCS, gentamicin (Life Technologies,
Grand Island, NY); RPMI 1640 containing 25 mM HEPES and
L-glutamine (BioWhittaker, Walkersville, MD); Percoll
(Pharmacia, Piscataway, NJ); Tris, Tween 20 (Bio-Rad, Hercules, CA);
leupeptin, DTT, PMSF (Boehringer Mannheim, Indianapolis, IN); rabbit
anti-phospho-extracellular signal-regulated kinase (ERK)
(mitogen-activated protein kinase (MAPK)) Ab and biotinylated molecular
mass markers (New England Biolabs, Beverly, MA); peroxidase-linked
donkey anti-rabbit Ig Ab and peroxidase-linked sheep anti-mouse
Ig Ab (Amersham, Arlington Heights, IL); PD98059 (Calbiochem, La Jolla,
CA); recombinant human IL-3 and recombinant human NGF (BioSource
International, Camarillo, CA); recombinant human IL-5 (R&D Systems,
Minneapolis, MN); and fura-2 acetoxymethyl ester (fura 2-AM; Molecular
Probes, Eugene, OR). Rabbit anti-cytosolic phospholipase
A2 (cPLA2). Ab was kindly
provided by Dr. L. Marshall (SmithKline Beecham, King of Prussia, PA).
Anti-phospho-Janus kinase 2 (JAK2), anti-phospho-STAT5,
anti-phosphotyrosine, 4G10, and Ras-binding domain (RBD; Raf-1
fragment)-Sepharose beads were obtained from Upstate Biotechnology
(Lake Placid, NY). Anti-JAK2, anti-STAT5 (G-2, binds to STAT5a and
b), anti-IL3R, anti-SHP2, anti-SHP1, and anti-SOS2
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-Grb2, anti-p21ras, and anti-Shc (both monoclonal and
polyclonal) were obtained from Transduction Laboratories
(Lexington, KY).
Buffers and media
PIPES-albumin-glucose (PAG) buffer consisted of 25 mM PIPES, 110 mM NaCl, 5 mM KCl, 0.1% glucose, and 0.003% HSA. PAGCM was PAG supplemented with 1 mM CaCl2 and 1 mM MgCl2. PAG-EDTA consisted of PAG supplemented with 4 mM EDTA. Countercurrent elutriation was conducted in PAG containing 0.25% BSA in place of 0.003% HSA. ESB is NOVEX electrophoresis sample buffer (NOVEX, San Diego, CA) containing 5% 2-ME. Complete lysis buffer (CLB) is 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 2 mM EGTA, 100 µg/ml aprotinin, 10 mM benzamidine, 5 mM DTT, 1 mM PMSF, 100 µg/ml leupeptin, 50 mM NaF, 5 mM Na4P2O7, 1 mM Na3VO4, and 1% Nonidet P-40. Incomplete lysis buffer is CLB without the protease inhibitors, Nonidet P-40 or vanadate. Stripping buffers were either 7 M guanidine hydrochloride or 65 mM Tris-HCl, 100 mM 2-ME, and 2% SDS. The sensitivity of the subsequent blotting to the choice of stripping agent determined which of these two were used.
Basophil purification
For most of these experiments, residual cells of normal donors undergoing leukapheresis were enriched in basophils using a combination of Percoll density gradients and countercurrent-flow elutriation, as previously described (20, 21). The cells were further purified by negative selection using a MACS basophil isolation kit (Miltenyi Biotec, Auburn, CA). More recently, we have used a mixture of Abs for negative selection from Stem Cell Technologies (basophil purification kit; Vancover, British Columbia, Canada) and columns from Miltenyi Biotec. The purity of basophils was determined by Alcian blue staining (22) and from these leukapheresis packs, generally exceeded 99%. For the basophil survival studies, basophils were isolated from fresh blood obtained by venipuncture. In these instances, the cells were first enriched for basophils using a double Percoll step gradient (1.066/1.079 g/ml) and purified to >90% using the Stem Cell reagents and Miltenyi columns as described above.
Basophils are the major, if not sole, leukocyte to release histamine and to generate LTC4 in response to a combination of cytokines (IL-3, IL-5, or NGF), and C5a, and IL-3 affects the basophil response directly (3, 4, 17, 23). We have previously confirmed that basophils were the major source of LTC4 release stimulated by IL-3 (for 15 min or 18 h) plus C5a (23) or by IL-5 or NGF under our experimental conditions (data not shown). Therefore, the basophil-enriched preparations used for the experiments where only histamine release and LTC4 generation were assessed had a somewhat lower mean purity (64 ± 17%; range, 47–99%).
Phosphorylation of ERKs and cPLA2
The phosphorylation of ERKs was assessed using an anti-phospho-ERK Ab and, in some early experiments, the electrophoretic mobility shift using anti-ERK1 and anti-ERK2 Abs as described previously (23, 24, 25). The phosphorylation of cPLA2 was assessed from the change in its electrophoretic mobility, as detected with anti-cPLA2 Ab (23, 26, 27). After basophils (1 x 106 cells/sample) were incubated with or without IL-3 in RPMI 1640 containing 2% FCS and gentamicin (25 µg/ml) at 37°C in a 5% CO2 incubator for the times indicated, reactions were stopped by adding ice-cold PAG (4x vol) and microfuged for 5–10 s. Cell pellets were immediately lysed in ESB. After boiling for 5 min, lysates were analyzed on Tris-glycine-polyacrylamide gels (NOVEX). After electrophoresis (160 V and 1.5 h for detection of ERKs phosphorylation, and 160 V and 3.5 h for cPLA2 phosphorylation), gels were then transferred to supported nitrocellulose membranes (OPTITRAN; Schleicher & Schuell, Keene, NH) with a Trans Blot (NOVEX). The membranes were immersed overnight in TBST containing 5% nonfat dry skim milk (Carnation, Los Angeles, CA). Immunoreactive proteins were detected using anti-phospho-ERK Ab or anti-cPLA2 Ab which were diluted in TBST containing 2% skim milk for 1.5 h. After washing, the membranes were incubated for 1 h with HRP-conjugated anti-rabbit Ab. After washing, membrane-bound anti-rabbit Ig Ab was visualized with ECL Western blotting detection reagents (Amersham) and Hyper-ECL luminescence detection film (Amersham). After detection by anti-phospho-ERK Ab, the same membranes were stripped with stripping buffer and reblotted with anti-ERK1 and anti-ERK2 Abs as described previously (23, 25).
Immunoprecipitation
After stimulating (cytokines or anti-IgE) basophils (1.5–5 x 106 cells/sample) in PAGCM buffer at 37°C, reactions were stopped by adding ice-cold PAG, or directly centrifuging for 5–10 s in an Eppendorf microfuge set for 16,000 x g. The cell pellets were immediately lysed in CLB. Lysates were precleared with protein G-Sepharose beads for 1 h at 4°C to remove any nonspecific binding to the beads. The lysates were then incubated with 1 µg/ml specific Ab prebound to protein G-Sepharose beads at 4°C. After a 1-h incubation, the beads were washed three times with CLB buffer. The immunoprecipitated proteins were eluted by boiling in ESB. Electrophoresis, transfer, and immunoblotting with anti-phosphotyrosine Ab (4G10) was performed as described above. The Ab was stripped from the membranes, and then membranes were reprobed with the indicated Abs.
Activated ras affinity precipitation assay
Activated ras affinity precipitation assay was performed as
described previously, with slight modifications (28, 29).
A GST fusion protein containing the RBD of raf-1 (aa 1–149 of raf-1),
which binds only GTP-bound (activated) ras, was immobilized on
glutathione-agarose beads (Upstate Biotechnology). After stimulating
basophils (
5 x 106 cells per condition),
reactions were stopped by adding ice-cold PAG and microfuged for 5–10
s. The cell pellets were immediately lysed in ras affinity
precipitation buffer (25 mM HEPES (pH 7.5), 2 mM EGTA, 150 mM NaCl, 10
mM MgCl2, 10% glycerol, 50 µg/ml aprotinin, 5
mM benzamidine, 50 µg/ml leupeptin, 25 mM NaF, 1 mM
Na3VO4, 1% Nonidet P-40,
and 1 mM PMSF). Clarified lysates were incubated with the GST-RBD beads
(5 µl/sample) for 1 h at 4°C with rocking. The GST-RBD beads
were washed three times with ras affinity precipitation buffer. Bound
proteins were eluted by boiling in ESB for 5 min. Affinity-precipitated
ras was detected by immunoblotting with anti-ras mAb.
DNA affinity adsorption for STAT5
DNA affinity purification was performed as described previously,
with modifications (30). Fc
R1-GAS probe
(5'-GTATTTCCCAGAAAAGGAAC) with 3'-terminal biotinylation and its
complementary strand were synthesized (BioSource International). After
annealing of the two single-strand oligonucleotides, the
double-stranded oligonucleotide was incubated with
streptavidin-conjugated agarose beads (Pierce, Rockford, IL) for 1
h at 4°C and washed twice with CLB buffer. After stimulating
basophils, the reaction was stopped by adding ice-cold PAG and cell
suspensions were microfuged. The cell pellets were immediately lysed in
CLB. Lysates were precleared with agarose beads for 1 h at 4°C
to remove any nonspecific binding to the beads. The lysates were then
incubated with GAS probe beads for 1 h at 4°C. The beads were
washed three times with CLB buffer and the affinity-adsorbed protein
was eluted by boiling in ESB for 5 min. Electrophoresis, transfer, and
immunoblotting was performed as described above.
Intracellular Ca2+ concentrations ([Ca2+]i) measurements
Basophils were labeled with 1 µM fura 2-AM for 20 min at 37°C in RPMI 1640 containing 2% FCS (300,000–500,000 cells in 200 µl; purity>90%). After washing once with 200 µl of PAG, the cells were resuspended in PAG for loading in the microscope observation chamber (16). [Ca2+]i changes were determined by digital video microscopy using techniques previously described in detail (16, 31). Briefly, 15 µl of cells (20,000–30,000) was loaded onto the siliconized coverslip of the microscope chamber and, after settling, overlaid with 1 ml of PAGCM buffer. After warming to 37°C, monitoring of the cells was begun and after four frames (each frame is a single ratio measurement of a field of 30–100 cells) of prechallenge [Ca2+]i levels were acquired, the cells were challenged with 1 ml of stimulus in buffer. Data were then acquired for 50–150 frames at intervals of 1–10 s to determine the subsequent [Ca2+]i response.
LTC4 measurements
Fifty thousand basophils were stimulated in a final volume of 100 µl of RPMI 1640 containing 2% FCS and 25 µg/ml gentamicin at 37°C in a 5% CO2 incubator. The reactions were terminated with 900 µl of ice-cold PAG-EDTA and the cells were then centrifuged in a microfuge at 14,000 rpm for 10 s. A RIA was performed using 100 µl of supernatant to determine LTC4 levels as previously described (1, 32). Each condition tested was performed in duplicate.
Flow cytometry
Basophil viability was assessed by staining with propidium iodide (2 µg/ml) for 1 min before analysis by flow cytometry. The percent positive cells (nonviable) was determined by setting a gate whose minimum value was at the point where >98% of unstained cells were excluded. The maximum of the gate was set to include the remaining range of fluorescence. Cells falling within this analysis region were deemed nonviable.
Real-time PCR
Purified basophils were cultured for 4 h with or without
cytokines and subjected to total RNA extraction using the RNAzol method
according to the manufacturer’s instructions. Samples were subjected
to reverse transcription and real-time PCR amplification with primers
specific to the human FcRI gene (33)
(5'-CCA-GGA-AGT-ATC-TTC-AGG-CAG-ACT-3';
5'-TCA-AAA-CTG-TCA-GCC-ATG-TAT-GC-3') along with an internal reporter
construct containing 6-carboxyfluorescein and
6-carboxytetramethylrhodamine fluorochromes
(5'-FAM-TTG-AAG-TCG-GCC-TCA-TCC-CCA-CC-TAMRA-3'; BioSource
International) using the TaqMan kit (PerkinElmer, Norwalk, CT).
Reaction conditions were as follows: reverse transcription at 48°C
for 30 min followed by PCR amplification (95°C for 10 min; 40 cycles
at 95°C for 15 s/60°C for 1 min). Real-time PCR monitors the time
course of the reaction by the appearance of unquenched fluorescence in
the sample tubes. The number of cycles required to obtain fluorescence
values 10 times the SD of the first 15 cycles of fluorescence was
calculated for each sample. Previous studies established that for this
probe and primer combination (using these reaction conditions), 2-fold
dilutions result in 1.02 ± 0.15 cycle differences. Therefore, the
relative expression of FcRI mRNA was calculated as 2^
(cycle difference between two samples).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IL-5 or NGF induce acute priming but not late priming.
As noted above, IL-3 primes basophils so that they secrete LTC4 when
stimulated with C5a. Treatment for 15 min or 24 h results in
changes in basophil behavior but the mechanisms for each phase are
different (23). Thus, we examined whether IL-5 or NGF
would also induce biphasic priming of basophils. IL-3-induced changes
were used as a positive control. As shown in Fig. 1
A, treatment with IL-5 or NGF
for 15 min significantly enhanced C5a-induced LTC4 release in the
concentration range of 1–100 ng/ml. The magnitude of LTC4 release in
cells stimulated with C5a was similar whether the cells were incubated
with IL-3 or IL-5, although NGF was slightly less effective, as
reported previously (3, 4). In contrast with the acute
effect, significant enhancement of C5a-induced LTC4 release was not
observed after 18-h pretreatment with IL-5 or NGF, whereas marked LTC4
release was caused by C5a from the cells treated with IL-3. The
magnitude of the acute effect for IL-5 (10–100 ng/ml) and IL-3 (10
ng/ml) was similar so we examined the relative potency of the two
cytokines to acutely prime the cell for LTC4 release. As shown in Fig. 2, the EC50 for the
priming effect by IL-3 was 
0.2 ng/ml, as reported previously
(23), whereas that for IL-5 was higher
(EC50 1 ng/ml).
|
|
RI.
We have recently reported that IL-3 induces marked changes in both mRNA
and protein for the subunit of the high-affinity IgE receptor
FcRI (34). We examined FcRI mRNA in basophils
stimulated with 10 ng/ml IL-3, IL-5, or NGF. Fig. 3 shows that both IL-3 and IL-5 induced
similar levels of mRNA while NGF had no effect. In one of these
experiments, we examined 30 and 100 ng/ml NGF, but these concentrations
were also without effect (data not shown).
|
. In pilot studies, earlier times were
examined, and the data were consistent with the data in Table I, but
the 3-day time point provided better discrimination. Notably, NGF was
the poorest at promoting survival, which in these studies was most
effectively measured by the viability of the cells rather than their
recovery. Cell recovery paralleled the viability but the difference
between medium alone and IL-3 was modest (60% of the cells placed in
culture where recovered in IL-3 cultures and 52% were recovered from
cultures of medium alone). An assessment of viability with either
erythrocin B or propidium iodide yielded similar results. Notably, the
difference in viability between NGF and medium alone was significant
for either method, p < 0.0001 and p =
0.033 for erythrocin B and propidium iodide, respectively. (IL-3 and
IL-5 were also statistically different from medium alone.)
|
ERK pathway.
Before doing comparative studies of the three cytokines, we first
examined in greater detail some of the signaling steps that could be
surmised from other cell models to follow stimulation with IL-3. In
pilot studies, we first examined tyrosine-phosphorylated proteins
following activation with IL-3, processed by generating whole-cell
lysates and Western blotting with anti-phosphotyrosine Ab. A
transient increase in tyrosine phosphorylation of several proteins
(130, 120, 95, 70, 55, 45, and 42 kDa) was observed (data not
shown). Reblotting with specific Abs suggested these proteins were ERK1
(p44 MAPK) and ERK2 (p42 MAPK), as described previously (23, 37), and IL-3R (c), JAK2, STAT5, SHP2, and Shc at 130,
120, 95, 70, and 55 kDa, respectively (data not shown).
With these results as a basis for further study, we examined these
signaling elements in greater detail using immunoprecipitation. From
extensive studies in other cell models, JAK2 phosphorylation of
IL-3R (c) and other cytoplasmic signaling proteins are likely to
be important in transmitting signals for cellular functions
(38). Not surprisingly, as shown in Fig. 4, A and B, both
JAK2 and IL-3 R were indeed tyrosine phosphorylated upon activation
with IL-3. A maximum phosphorylation of JAK2 and IL-3 R was observed
at 5 min and had decreased to near basal levels by 45 min.
|
c (by IL-3, IL-5, and GM-CSF) that leads to JAK2 activation results
in the phosphorylation of Shc (38). Phosphorylated Shc can
then bind to Grb2/Sos dimers to recruit them to the cell surface. The
GDP/GTP exchange factor, Sos, in the assembled complex interacts with
p21ras and initiates the swap of GDP for GTP in
ras. Since we have previously demonstrated that acute enhancement of
C5a-induced LTC4 release from basophils requires ERK and
cPLA2 phosphorylation (25, 27), an
examination of these elements was warranted. Thus, phosphorylation of
Shc and association of Grb2 and Shc induced by IL-3 were examined. As
shown in Fig. 4
C, Shc was also transiently phosphorylated by
exposure of the cells to IL-3. Similar to phosphorylation of c and
JAK2, a maximum phosphorylation of Shc was observed at 5 min and had
decreased to near basal levels by 45 min. To determine whether Grb2 and
Shc were associated following stimulation, Grb2 was immunoprecipitated
from cell lysates and subsequent Western blotting was performed with
anti-Shc. Tyrosine-phosphorylated 52- and 70-kDa proteins were
coimmunoprecipitated by anti-Grb2 Ab (Fig. 4D) and their
presence was transitory, i.e., present at 5 min and not present after
45 min. The tyrosine-phosphorylated 52- and 70-kDa bands were found to
colocalize with bands following blotting with anti-Shc and
anti-SHP2 Ab, respectively. These results suggest that both
phosphorylated proteins were associated with Grb2. To verify the
tyrosine phosphorylation of SHP2, immunoprecipitation with
anti-SHP2 Ab and immunoblotting with anti-phosphotyrosine Ab
was performed. As shown in Fig. 4E, SHP2 was also
transiently phosphorylated following stimulation with IL-3. It should
be noted that SHP1 was not detected in the proteins immunoprecipitated
with anti-Grb2 and tyrosine phosphorylation of SHP1 following
activation with IL-3 was not detected (although SHP1 is expressed in
basophils (39), and data not shown).
Next, we examined activation of p21ras using
affinity precipitation by a GST fusion protein containing the RBD of
raf, since activated ras protein (ras-GTP) bound to RBD (see details in
Materials and Methods). As shown in Fig. 4F, the
mass of ras (GTP-bound form) was markedly increased following
activation with IL-3. This result also demonstrates that activation of
p21ras following stimulation with IL-3 was
transient. Although our previous studies examined phosphorylation of
ERKs (23), we verified that ERK phosphorylation was also
transient in the current studies (data not shown).
STAT5/JAK2 pathway.
Phosphorylation of STAT5 (by JAK2) is likely to be important in
transmitting signals from the cell surface to the nucleus
(38). Therefore, IL-3-induced phosphorylation of STAT5 was
examined. As shown in Figs. 5A
and 6, STAT5 was phosphorylated following
activation with IL-3, peaking from 5 to 15 min. This phosphorylation
was decreased to 46% of maximum by 45 min, suggesting that activation
of STAT5 is more sustained than that of JAK2 but nevertheless transient
relative to some of the 24-h or multiday priming effects we observe
with IL-3. These kinetics apply whether the cells are stimulated in
PAGCM or RPMI 1640 (+2% FCS; data not shown). It is worth noting that
this difference in behavior between JAK2 and STAT5 kinetics (Fig. 6)
also translates to the concentration dependence of the response. Fig. 5C shows that stimulation with 1 ng/ml IL-3 results in a
response that is <10% of the response at 10 ng/ml for both
phosphorylation of IL-3 receptor and JAK2 while the phosphorylation
of STAT5 at 1 ng/ml is 80% of the response observed using a
concentration of 10 ng/ml. This concentration dependence varies
significantly among basophil preparations but the relative relationship
between JAK2 and STAT5 phosphorylation persists. Activated
(phosphorylated) STAT5 dimerizes via association of one partner’s SH2
domain to the other partner’s phosphorylated tyrosine residue. The
dimer, in turn, gains the ability to bind to DNA. The ability of STAT5
to bind to a relevant DNA oligonucleotide was examined using the GAS
probe (a nucleotide sequence bound by most of the STAT family of
proteins). As shown in Fig. 5B, tyrosine-phosphorylated
STAT5 was detected in samples prepared using DNA affinity precipitation
or STAT5 immunoprecipitation. However, STAT5 protein was only detected
with DNA affinity precipitation following activation with IL-3,
suggesting that phosphorylated STAT5 is capable of binding to DNA. Fig. 6 summarizes the kinetic results for some of these signaling
elements.
|
|
A, phosphorylation of JAK2,
Shc, STAT5, and ERKs induced by IL-3 was inhibited by staurosporine but
not Ro-31-8220. In data not shown, 1 µM staurosporine also inhibited
IL-3-induced expression of FcRI mRNA. Not shown in the figure are
experiments which indicate that the putative JAK2 kinase inhibitor
AG490 does not alter either JAK2 or STAT5 phosphorylation. Fig. 7B demonstrates that stimulation with IL-3 does not cause
phosphorylation of syk and that SHP2 phosphorylation occurs with IL-3
stimulation but not anti-IgE Ab. We have previously demonstrated
that activation of the ERK pathway following stimulation with
anti-IgE Ab is sensitive to the phosphatidylinositol 3-kinase (PI3)
inhibitor LY294002. The results of IL-3-induced ERK phosphorylation in
the presence of LY294002 have been variable. Fig. 7C shows
that there was little or no inhibition of ERK phosphorylation by 10
µM LY294002 (a concentration which completely inhibited IgE-mediated
ERK phosphorylation). But, we have found 50% inhibition of
IL-3-mediated ERK phosphorylation in the presence of 30 µM LY294002
(data not shown). Wortmannin also had only modest effects. However, we
have not noted any inhibition of IL-3-induced STAT5 phosphorylation by
LY294002, even at 30 µM (Fig. 7C). The putative lyn
kinase inhibitor, PP1, effectively inhibited ERK phosphorylation
induced by anti-IgE Ab (as shown previously) but not IL-3 (Fig. 7D).
|
ERK pathway.
The above studies indicate that IL-3 signals
through at least two pathways that regulate transcription factors, one
which leads to ERK phosphorylation (and cPLA2
phosphorylation)—the shc/Grb2/SOS/ras/ERK pathway—and one which
results in STAT5 activation. We first examined phosphorylation of ERKs
and cPLA2 in response to IL-5 or NGF, as these
were the end points of the relevant pathway resulting in enhanced LTC4
release. Phosphorylation of ERKs was assessed by immunoblotting with
anti-phospho-ERKs Ab and phosphorylation of
cPLA2 was assessed using the shift in
electrophoretic mobility as detected with
anti-cPLA2 Ab, as previously described
(23, 25). IL-3-induced phosphorylation of ERKs and
cPLA2 was used as a positive control and the time
of incubation was previously shown to be optimal for IL-3
(23). As shown in Fig. 8, A and B, IL-5 or NGF induced phosphorylation of
ERKs and of cPLA2 at levels that were similar to
those induced by IL-3. Changes in the electrophoretic mobility of ERK1
(data not shown) and ERK2 supported the phospho-ERK blot data. ERK2
reblotting also demonstrated essentially equal protein loading for each
lane because the sum of phosphorylated and nonphosphorylated forms was
equal in all lanes. We have found that on average, at the 5-min time
point and using 10 ng/ml of each of the cytokines, that IL-5 and NGF
induce 0.56 ± 0.15 (n = 6) and 0.45 ± 0.20
(n = 9) of the phosphorylation observed following IL-3,
respectively. The range of the latter ratio (NGF:IL-3) is marked, with
a low of 0.05 and a high of 2.0. Not surprisingly, both IL-3 and IL-5
caused phosphorylation of Shc (data not shown). NGF also induced
phosphorylation of Shc (Fig. 8B). These results suggest that
activation of ERKs and cPLA2 may be responsible
for the acute priming effect for LTC4 generation induced by IL-5 or
NGF. Although the magnitude of ERK1/2 phosphorylation was similar for
IL-3 and IL-5, the kinetic profile was somewhat different. As shown in
Fig. 8C, IL-5- and NGF-induced ERK1/2 phosphorylation is
more transient than that induced by IL-3. By 30 min, IL-5-induced
ERK1/2 phosphorylation had returned to the basal level whereas
IL-3-induced phosphorylation was still observed. Consistent with the
kinetics of phosphorylation of ERK1/2, the priming effect of IL-5, as
assessed by enhanced C5a-induced LTC4 release, was also more transient
than that caused by IL-3 (Fig. 8D). Data in Table II show that in basophils primed with all
three cytokines, PD98059 (a MAPK kinase inhibitor) inhibited
C5a-induced LTC4 release.
|
|
subunit, both
IL-3 and IL-5 also activate JAK2 and STAT5 (Fig. 9, A and B).
However, NGF does not appear to activate either JAK2 or STAT5 (Fig. 9, C and D). It is apparent that although NGF is
capable of activating the ERK pathway, it often does so less
effectively than IL-3. We considered the possibility that IL-3 appeared
to induce phosphorylation of STAT5 because it generates a much stronger
signal than NGF. To compare the relative strength of the signals, we
titrated the IL-3 response with serial dilutions of IL-3 so that the
ERK response matched the lesser ERK response induced by NGF. At this
point of equivalence (generally occurring at 0.3 ng/ml IL-3), IL-3
continued to induce significant phosphorylation of STAT5 (data not
shown).
|
, treatment of IL-5 or NGF for
18 h did not alter C5a-induced
[Ca2+]i, whereas IL-3
markedly enhanced C5a-induced
[Ca2+]i.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI while NGF did not. Third, IL-3 was best at maintaining the
viability of basophils in culture, with IL-5 and NGF allowing better
viability than medium alone but significantly less than with IL-3. Of
these observations, the most interesting dichotomy for two cytokines
that were capable of priming basophils for C5a-induced LTC4 release was
between IL-3 and NGF and the marked difference in their ability to
induce mRNA accumulation for FcRI. It was in this context that we
were most interested in the signaling differences for the two
cytokines. The expectations for signaling with IL-5 were that it would
be a weaker version of the signaling for IL-3, since these two share a
common subunit and it is known that there may be up to 10-fold
fewer IL-5 subunits expressed on the basophils surface than IL-3
(40). The difference in potency between IL-3 and IL-5 and
the otherwise similar affinity of the two cytokines for their
respective receptors suggests that any quantitative difference in
signaling for these two cytokines is a reflection of the differences in
receptor density. An important caveat to this statement is our lack of
knowledge concerning the quality of the recombinant cytokine
preparations. It is unclear whether mole for mole, the commercial
preparations of these two cytokines could be considered equal in
ability to bind to their respective subunits. For example, we have
found that there is significant variation in the potency of IL-3
preparations even from the same manufacturer. However, under the best
of conditions for both IL-3 and IL-5, there continues to be a
difference in potency.
To understand the origins of the differences between IL-3 and NGF, we
first examined some of the signaling steps well described in studies of
cell lines. Because studies which examine cause and effect for
signaling elements in human basophils are quite limited, it is
necessary to interpret the basophil studies in light of what is known
from studies of cell lines. The expectation was that IL-3 binding to
its receptor and the subsequent receptor dimerization would result in
activation of JAK2 kinase, most likely due to JAK2
transphosphorylation. JAK2 phosphorylation of IL-3R (c) and other
cytoplasmic signaling proteins (Shc and STAT5) is likely to be
important in transmitting signals for cellular functions
(38). In this context, we found expected changes in these
signaling elements in human basophils. If the cell line models are
valid for human basophils, our results suggested that phosphorylation
of JAK2 and Shc occurred, followed by association of Shc and Grb2 and
ras activation leading to ERK activation for the priming effect that
acutely alters LTC4 release. These activation events were transient and
kinetically associated with ERK activation. These transient
characteristics are then reflected in the transient acute priming by
IL-3 for LTC4 release, as documented in previous studies
(23). It seems likely that activation of TrkA receptors on
basophils, by NGF also results in the phosphorylation of Shc by the
intrinsic kinase activity of the TrkA receptor (6), which
results in activation of the ERK pathway that is similar to what is
observed with IL-3. NGF-mediated activation of the ras-ERK pathway has
been reported in other cells (41, 42, 43). The strength of the
initial signals is, on average, weaker for NGF and, like IL-5, the
weaker activation results in a more transient response, both at the
level of ERK phosphorylation and priming for C5a-induced LTC4
release.
An interesting aspect of the JAK2/STAT5 response is that a low JAK2
signal translates to a stronger STAT5 signal. This can be observed in
the dose-response curve for IL-3, the comparison of IL-3 and IL-5, or
the longevity of the STAT5 response relative to the JAK2 response. The
data have the appearance of a signaling element that integrates the
strength of the antecedent signal. This may come about because the
opposing dephosphorylation reaction for STAT5 is weak or the STAT5
translocates out of its normal environment, something that would not be
surprising for a transcription factor. It has been reported that JAK2
activation and its downstream events are susceptible to staurosporine
(a general protein kinase inhibitor) (44, 45). In human
basophils, staurosporine also inhibited phosphorylation of JAK2 and its
putative downstream signaling events (phosphorylation of Shc, STAT5,
and ERKs). In contrast, Ro-31-8220 (PKC-specific inhibitor) did not
affect these events, indicating that a PKC is not responsible for these
events. AG490 (tyrphostin B42) has been reported as a JAK2 inhibitor
but we could not find evidence that it acted on JAK2 using
concentrations as high as 100 µM. With respect to ERK
phosphorylation, it is less clear how involved is a PI3 kinase since
the selective inhibitor, LY294002, had variable and incomplete effects,
and the sporadic inhibition observed only occurred at relatively high
concentrations. Significant inhibition of IgE-mediated secretion and
ERK phosphorylation occurs at 3–4 µM (IC50).
On the other hand, we have also previously found that somewhat higher
concentrations of LY294002 (an IC50 of 10
µM) inhibit the IgE-mediated elevation in cytosolic-free calcium.
Nevertheless, the high concentrations required to observed any
inhibition suggest possible nonselective effects of the drug. In
contrast, LY294002 did not inhibit STAT5 phosphorylation, suggesting
that this pathway is unaffected by PI3 kinase activity, a result that
is perhaps not surprising given the relatively direct connection
between JAK2 activation and STAT5 phosphorylation. Finally, although
there are indications that lyn kinase plays a role in IL-5 priming of
eosinophils (46), we could not detect an effect of PP1 on
either the ERK or STAT5 pathways. In contrast, we have shown that it
effectively inhibits a variety of IgE-mediated functions and signaling
elements (47), data which are consistent with its ability
to inhibit lyn kinase.
Phosphorylation of Shc is a critical step for ras activation since phosphorylated Shc can then bind to the Grb2-Sos complex to recruit them to the cell surface where ras is located. The IL-3-induced transient activation of the ras-ERK pathway can be explained by the transient phosphorylation of Shc (or the association of Shc to Grb2) rather than the possibility that a dissociation of Sos from Grb2 occurs (which has been reported to occur in other cell types under certain conditions (48, 49)). We have previously demonstrated that human basophils express Sos2 but not Sos1 and that Sos2 protein is constitutively associated with Grb2.4 Sos2 protein is not phosphorylated or dissociated from Grb2 following activation with IL-3 in human basophils (data not shown). It has been reported that the role of SHP2 in cytokine receptor activation is context sensitive, in some cases acting as a positive regulator of cytokine signaling (50) and sometimes acting as a negative regulator (51). We have shown that SHP2 appeared to be phosphorylated on tyrosine in response to IL-3 and associated with Grb2 but have not determined whether this has a positive or negative influence on the course of the signaling events.
In contrast with the relative similarity of acute priming events, there
were marked differences in the effects of these three cytokines on
responses that could be considered to require a longer cascade of
events. Table III summarizes these
differences as well as the differences in signaling. Unlike the ability
of IL-3 to augment the
[Ca2+]i response that
follows stimulation with C5a, and in particular to enhance the second
phase of the [Ca2+]i
response (15, 23), treatment with IL-5 and NGF was similar
to culture without any cytokines. These results also support the
hypothesis that the sustained (or enhanced)
[Ca2+]i response
facilitated by IL-3 is essential for its late priming effect
(23). The absence of a late priming effect using NGF might
not be surprising since the TrkA receptor may initiate signaling that
is different from the signaling started by the subunit of
IL-3/IL-5. Indeed, these studies show that there is at least one
signaling difference between the IL-3 receptor and TrkA receptors, the
absence of JAK2 and STAT5 activation by NGF. However, IL-3 and IL-5
presumably operate through the same subunit. This difference in
JAK2 activation and the more transient nature of IL-5 acute priming
suggests that the difference at 18 h may be the result of
quantitative issues rather than qualitative aspects of signaling.
|
RI compared with NGF is
also the result of the inability of NGF to induce STAT5 activation.
Unfortunately, we have yet to find a specific inhibitor that would
allow us to demonstrate that FcRI mRNA accumulation requires
STAT5 activation. We did find that staurosporine inhibited both
IL-3-induced STAT5 phosphorylation and FcRI mRNA up-regulation,
but this drug is so broad a kinase inhibitor that this result is
difficult to interpret. An intriguing observation that places NGF in a
somewhat different category is that it does alter basophil viability in
a relatively long-term culture, albeit much less so than IL-3. In some
experiments, IL-5 and NGF resulted in a similar retention of viability,
indicating the close similarity in the effect of these two cytokines.
To reflect this similarity, we assigned a single plus for both IL-5 and
NGF in this category in Table III and a double plus for IL-3.
Considering that there was a reasonable association between the
activation of STAT5 and the ability of a cytokine to up-regulate
FcRI mRNA, the partial dissociation between STAT5 activation and
survival (IL-3 vs NGF) is notable. When the data are viewed from the
crude perspective of the plus-minus assignments in Table III, it
appears that acute LTC4 priming and survival match ERK activation while
FcRI mRNA up-regulation matches STAT5 activation. However, the
overnight enhancement of the cytosolic calcium response matches neither
signaling pathway. It remains possible that the enhancement of the
calcium response depends on ERK pathway activation but is more
sensitive than survival to the relative strength of this activation.
Alternatively, survival depends not on ERK activation but pathways not
yet studied.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Donald W. MacGlashan, Jr., Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail address: dmacglas{at}welch.jhu.edu
3 Abbreviations used in this paper: LTC4, leukotriene C4; ERK, extracellular signal-regulated kinase; NGF, nerve growth factor; HSA, human serum albumin; MAPK, mitogen-activated protein kinase; JAK2, Janus kinase 2; PAG, PIPES-albumin-glucose; CLB, complete lysis buffer; cPLA2, cytosolic phospholipase A2; [Ca2+]i, intracellular Ca2+ concentration; RBD, Ras-binding domain; PKC, protein kinase C; PI3, phosphatidylinositol 3-kinase.
4 K. Miura, S. E. Lavens-Phillips, and D. W. MacGlashan, Jr. Localizing a control point in the pathway to LTC4 secretion following stimulation of human basophils with anti-IgE antibody. Submitted for publication.
Received for publication March 9, 2001. Accepted for publication June 8, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain and expression of the complete human receptor. J. Biol. Chem. 267:12782.RI and FcRI in human blood basophils. J. Allergy Clin. Immunol. 107:832.[Medline]
RI expression on human basophils by IgE antibody. Blood 91:1633.RI signaling by FcRII costimulation in human blood basophils. J. Allergy Clin. Immunol. 106:337.[Medline]
This article has been cited by other articles:
|
|
 |
|
  L. Pieri, C. Bogani, P. Guglielmelli, M. Zingariello, R. A. Rana, N. Bartalucci, A. Bosi, and A. M. Vannucchi The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera Haematologica, November 1, 2009; 94(11): 1537 - 1545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Pecaric-Petkovic, S. A. Didichenko, S. Kaempfer, N. Spiegl, and C. A. Dahinden Human basophils and eosinophils are the direct target leukocytes of the novel IL-1 family member IL-33 Blood, February 12, 2009; 113(7): 1526 - 1534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Didichenko, N. Spiegl, T. Brunner, and C. A. Dahinden IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils Blood, November 15, 2008; 112(10): 3949 - 3958. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Spiegl, S. Didichenko, P. McCaffery, H. Langen, and C. A. Dahinden Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid Blood, November 1, 2008; 112(9): 3762 - 3771. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. Vonakis, D. W. MacGlashan Jr, N. Vilarino, J. M. Langdon, R. S. Scott, and S. M. MacDonald Distinct characteristics of signal transduction events by histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced priming and activation of human basophils Blood, February 15, 2008; 111(4): 1789 - 1796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. MacGlashan Jr. Endocytosis, recycling, and degradation of unoccupied Fc{epsilon}RI in human basophils J. Leukoc. Biol., October 1, 2007; 82(4): 1003 - 1010. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Tschopp, N. Spiegl, S. Didichenko, W. Lutmann, P. Julius, J. C. Virchow, C. E. Hack, and C. A. Dahinden Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma Blood, October 1, 2006; 108(7): 2290 - 2299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Vilarino, K. Miura, and D. W. MacGlashan Jr Acute IL-3 Priming Up-Regulates the Stimulus-Induced Raf-1-Mek-Erk Cascade Independently of IL-3-Induced Activation of Erk J. Immunol., September 1, 2005; 175(5): 3006 - 3014. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Sloane, N. Tedla, M. Awoniyi, D. W. MacGlashan Jr, L. Borges, K. F. Austen, and J. P. Arm Leukocyte immunoglobulin-like receptors: novel innate receptors for human basophil activation and inhibition Blood, November 1, 2004; 104(9): 2832 - 2839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Iikura, M. Ebisawa, M. Yamaguchi, H. Tachimoto, K. Ohta, K. Yamamoto, and K. Hirai Transendothelial Migration of Human Basophils J. Immunol., October 15, 2004; 173(8): 5189 - 5195. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), IL-5 (10 ng/ml, O), or
NGF (10 ng/ml, 
) for 18 h, washed, and labeled with fura-2 AM.
After washing, the cells were stimulated with C5a (50 ng/ml). The
[Ca2+]i response was analyzed as described in
Materials and Methods. The results shown are
representative of two separate experiments.