Johns Hopkins University, Asthma & Allergy Center, Baltimore, Maryland
Correspondence: Donald MacGlashan Jr., M.D., Ph.D., Johns Hopkins University, Asthma & Allergy Center, 5501 Bayview Circle, Baltimore, MD 21224. E-mail: dmacglashan{at}welch.jhu.edu
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Key Words: mediator release • histamine release • protein kinase C • syk and lyn kinases
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Our interest in developing a better understanding of the characteristics of this quiescent period was piqued by observations about the length of the quiescent period (what we call the timelag of the [Ca++]i response) in basophils that have been called nonreleasers (the observations of which will be presented) [5 , 6 ]. Nonreleasing basophils have been shown to possess cell-surface densities of IgE and receptor that are equivalent to releasing basophils but do not release mediators in response to stimulation with anti-IgE antibodies, anti-receptor, antibodies, or antigens (on cells passively sensitized with antigen-specific IgE) [5 ]. It can be shown that these aggregating stimuli bind to the cell surface and even induce changes in basophil function that indicate that at least a partial signal has occurred. Understanding the causes of this phenotype may lead to an appreciation of processes that regulate the expression of basophil function. The precise nature of the defect is not yet fully understood, although recent studies have suggested that these cells are deficient in the expression the tyrosine kinase, p72syk [7 , 8 ]. A variety of studies in other related cell types have demonstrated the critical importance of p72syk as an early tyrosine kinase in the IgE-mediated signaling cascade [9 10 11 12 13 14 15 16 17 18 ], and its absence could explain the nonreleaser phenotype. However, there are aspects to the [Ca++]i response in nonreleasing cells that raise questions about whether such a deficiency can fully explain this phenotype. Furthermore, the characteristics of the [Ca++]i response in nonreleasing cells also raise questions about the factors regulating the quiescent (timelag) period of the [Ca++]i response. The following studies were designed to characterize this quiescent period using a pharmacological approach and then to explore the characteristics of the response in releasing and nonreleasing basophils.
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Buffers
PIPES buffer contained 25 mM PIPES, 110 mM NaCl, and 5 mM KCl,
adjusted to pH 7.4 with 1 N HCl; PIPES-albumin-glucose (PAG) contained
0.003% (w/v) HSA and 0.1% (w/v) glucose; PAGCM was PAG with 1 mM
CaCl2 and 1 mM MgCl2; and
elutriation buffer for the purification of human basophils contained
PIPES buffer, 0.1% (w/v) glucose and 0.25% (w/v) BSA. Lysis buffer
contained 20 mM Tris (pH 7.8), 150 mM sodium chloride, 1% NP-40, 5%
glycerol, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1
mM benzamidine, and 1 µg/ml aprotinin. In the electrophoresis
studies, 2x sample buffer contained 0.5 M Tris-HCl, pH 6.8, 10% (w/v)
SDS, 0.1% bromophenol blue, 20% glycerol, and 5% mercaptoethanol;
TBST buffer contained 12 mM Tris base (pH 7.5), 150 mM NaCl, and 0.05%
Tween-20; running buffer contained 25 mM Tris base, 192 mM glycine, and
0.1% SDS; transfer buffer contained 12 mM Tris base, 96 mM glycine,
and 20% methanol; and stripping buffer contained 7 M guanidine
hydrochloride.
Isolation of human basophils
Basophils were purified from buffy coat cells obtained from
normal donors undergoing leukapheresis as previously described
[21
]. The leukocytes were partially purified by Percoll
density gradients and counter-current elutriation. The basophils were
then placed into culture (RPMI 1640 with 2% FCS and 20 µg/ml
gentamicin) for 1 h after elutriation and then further purified on
a subsequent, two-step Percoll density gradient (1.066/1.079). After
culturing overnight in RPMI 1640 supplemented with 2% FCS, gentamicin
and in some later experiments, 30 pg/ml interleukin (IL)-3 (a
concentration pre-determined to only maintain better viability but with
no measurable priming effect on basophil function). For most of
these experiments, the cells were purified further by an additional,
two-step Percoll gradient (1.0067/1.079 g/ml). However, in more recent
experiments, the cells were purified further by negative selection
using Miltenyi reagents and columns (Miltenyi Biotec Gmbh). We have not
detected a difference in the behavior of the cells purified by the two
methods. For these studies, basophil purities were >95% and most
often nearly 100%.
For the studies of releasers and nonreleasers, donors consisted of adult subjects who were selected from the personnel of the Department of Clinical Immunology. The donors were nonatopic as assessed by the questionnaire. For the purpose of this study, histamine release from human basophils in response to anti-IgE (0.2 µg/ml) antibody was determined on at least three different occasions. A "nonreleaser" was defined as a donor whose basophils released <5% histamine, and a "releaser" was a donor whose basophils released >10% histamine on all three occasions. Donors were asked if they had any medical problems, especially any known history of atopy (asthma, allergic rhinitis, chronic urticaria, atopic dermatitis, etc.), and if they were receiving any medication. Blood was drawn by venipuncture, and the cells were separated using a two-step, Percoll density gradient. Briefly, the leukocytes were partially purified in accuspin tubes on a two-step, Percoll density gradient (1.070/1.082 g/ml). The interface containing the basophils (10–20% pure) was removed and washed in PAG. The basophils were then further purified by negative selection using the Miltenyi basophil isolation kit (which contains a cocktail of hapten-conjugated CD3, CD7, CD14, CD15, CD16, CD36, CD45RA, and HLA-DR antibodies and MACS microbeads coupled to an anti-hapten-conjugated mAb) and glycophorin A microbeads. Any remaining contaminating cells consisted mainly of monocytes and lymphocytes. This procedure required 2–3 h of preparation, and purities ranged 90–98%.
Cell counting
Basophils were stained with Alcian blue [22
] and
counted in a Spiers-Levy hemocytometer.
Histamine release and sensitization
All reactions were carried out in PAGCM buffer. Cell
concentration depended on the assay; simple histamine release used
2 x 104/ml, and assessment of syk phosphorylation
used 7.5–10 x 106 basophils/ml. For the kinetics of
histamine release, the reactions were stopped with ice-cold PAG buffer
containing 10 mM EDTA. The supernatants were harvested after
centrifugation. In each experiment, perchloric acid at a 1.6% final
concentration was added to some tubes to determine total histamine
content. Histamine was assayed by an automated fluorometric technique
[23
]. The percentage of histamine release was calculated
from the ratio of sample to total histamine after spontaneous release
was subtracted from both. For sensitization with BPO-specific IgE, the
cells were incubated for 25 min at 37°C with 5 µg/ml purified,
BPO-specific IgE in RPMI medium containing 10 µg/ml heparin and 1 mM
EDTA. Cells were used after three washes with PAG buffer. Previous
studies established that the 5 µg/ml concentration of BPO-specific
IgE was just sufficient to fully sensitize available unoccupied Fc
RI
during the time used for passive sensitization. Previous studies also
demonstrated that measured cell-surface density of BPO-specific IgE
roughly titrated according the concentration of BPO-specific IgE used
for sensitization [24
].
Immunoprecipitation
For syk phosphorylation studies, 1.5–2 x
106-purified basophils/condition were resuspended in PAGCM
and stimulated with anti-IgE antibody at a final concentration of 0.2
µg/ml. At various times after stimulation, the cell suspension was
centrifuged at 14,000 g for 10 s, the supernatant was
removed, and the lysis buffer (1 ml) was added to the cell pellet.
After sitting on ice for 10 min, the tubes were centrifuged for 3 min
at 14,000 g. Centrifuged lysates were pre-cleared by
incubation with protein G sepharose beads for 30 min at 4°C. The
pre-cleared lysates were then incubated with 1 µg/ml anti-p72syk
pre-bound to protein G sepharose beads. After gentle rotation for
1 h at 4°C, the beads were washed, and the immunoadsorbed
proteins were eluted from the beads by boiling in 2x SDS sample
buffer. Control experiments revealed that an irrelevant IgG antibody or
mouse anti-human p72syk in the absence of lysate did not pull down syk
in the immunoprecipitates.
Blotting of proteins
Proteins were separated in 10% tris-glycine gels under reducing
conditions and electrotransferred on to a nitrocellulose membrane. The
free binding sites were blocked by incubating the membrane overnight at
4°C with 3% BSA in TBST. The nitrocellulose membranes were then
incubated with 0.5 µg/ml anti-phosphotyrosine mAb, 4G10, in 1%
BSA/TBST for 1 h at room temperature. The membrane was then washed
with TBST prior to the addition of an anti-mouse HRP conjugate (1:3000
dilution) for 1 h at room temperature. After further washing of
the membranes with TBST, the phosphoproteins were visualized using ECL.
The nitrocellulose membrane was exposed to ECL hyperfilm for 15 s–10
min. Following exposure, the nitrocellulose membrane was stripped for
30 min at room temperature with stripping buffer and re-probed with 0.2
µg/ml mouse anti-human p72syk. After exposure to
chemiluminescence-detection agents, the intensity of each band was
determined using densitometric analysis using a Kodak DC120 digital
camera and acquisition software. In pilot experiments, we determined
from twofold dilutional analysis that ECL detection was linear over the
range of detection required.
[Ca++]i measurements
Basophils were labeled with 1 µM fura-2AM for 20 min at 37°C
in RPMI 1640 containing 2% FCS (300,000–500,000 cells in 200 µl).
After washing once with 200 µl PAG, the cells were resuspended in PAG
for loading in the microscope stage [25
,
26
]. [Ca++]i was
determined by digital video microscopy using techniques previously
described in detail [25
, 27
]. Briefly, 15
µl cells (20,000–30,000) were loaded onto the siliconized coverslip
of the microscope chamber and, after settling, overlaid with 1 ml PAGCM
buffer. After warming to 37°C, monitoring of the cells was begun and
after several frames (each frame is a single-ratio measurement of a
field of 30–100 cells) of pre-challenge
[Ca++]i levels were acquired, the
cells were challenged with 1 ml stimulus in buffer. Data were then
acquired for 50–150 frames at intervals of 1–10 s to determine the
subsequent [Ca++]iresponse. For studies of basophil response in the microscope
chamber (resting on the siliconized coverslip), the concentration of
anti-IgE antibody that is optimal for histamine release occurs at a
slightly higher concentration than is typical for experiments in test
tubes (0.5 µg/ml vs. 0.2 µg/ml, respectively). The
[Ca++]i timelag is the difference
between the time of stimulus addition and the first statistically
significant elevation in [Ca++]i.
(See Figure 2A for an example of two single cells and their associated
timelag intervals.)
Statistics
For the measurements of net average
[Ca++]i increase and the
[Ca++]i timelag, the errors noted
in Table 1
represent the errors associated with averaging the results from
separate experiments (where the net elevation in
[Ca++]i for any one condition is
itself an average of 30–70 cells), which we think better reflects the
biological variation inherent in using basophils from different donors.
Error bars in other plots represent the SE.
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Table 1. Effect of pharmacological agents known to inhibit the IgE-mediated cytosolic calcium response in human basophils on the timelag characteristic
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Figure 1. Differences in the Ca timelag in basophils stimulated with bivalent
penicillin (BPO2) or multivalent penicillin [BPO(11.5)-HAS].
Sensitized basophils were challenged with optimal concentrations of
BPO2 (50 nM;  ) or BPO(11.5)-HSA (0.5 µg/ml,  1 nM; •). (A)
Relative histamine release response for cells sensitized with serial
twofold dilutions of BPO-specific IgE. (B and C) Frequency histograms
for the [Ca++]i timelags for
fura-2-labeled cells stimulated with BPO(11.5)-HSA or BPO2,
respectively.
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RI. Others have demonstrated that PP1 and PP2 are potent
inhibitors of lyn kinase [33
, 34
], and we
found that either drug effectively inhibits phosphorylation of lyn and
syk kinases during stimulation of basophils with anti-IgE antibody
[35
]. Figure 2A
shows the effects of 0.8 µM PP1 on the timelag in basophils
stimulated with anti-IgE antibody. In data not shown, PP1 at 10 µM
had no effect on the characteristics of calcium response following
stimulation with formyl-Met-Leu-Phe (fMLP; which operates through a
G-protein-linked receptor). Figure 3
shows the frequency histogram of the cytosolic calcium response
timelag for all the cells examined. The timelag was increased
approximately fourfold at this concentration of PP1. It was remarkable
that the timelag could be extended to as long as 10 min with PP1 at
this concentration. Despite these long timelags, the ultimate response
appeared otherwise normal for most cells. Not as evident in the
single-cell traces, the average calcium response late in the reaction
was equivalent or greater than in cells not treated with PP1. For the
two experiments that form the basis of the histograms in Figure 3
, the
net cytosolic calcium elevation from 5–10 min averaged 33 ± 2 nM
for cells not treated with PP1 and 38 ± 6 nM for cells treated
with PP1, and the average (see Table 1 ) for the entire period of
observation was lower for the cells treated with PP1 because of the
delayed onset of the response. We also noted that at concentrations
below 1 µM, the kinetics of histamine release reflected the fact that
the calcium response was delayed but otherwise normal, i.e., at 0.8
µM PP1, and release did not occur for 8 min compared with 2 min in
cells not treated with PP1 but then progressed to the same final amount
of histamine release as the untreated cells (Fig. 2B)
. It should be
noted that the average timelag could be adjusted over a large range by
adjusting the concentration of PP1. Concentrations above 1 µM
resulted in many cells not responding within a timeframe of 15–20 min.
Figure 2C
also demonstrates that phosphorylation of syk kinase was
delayed and blunted in cells treated with 0.8 µM PP1.
 View larger version (32K): [in a new window] |
Figure 2. Effect of PP1 on the basophil calcium and histamine release. (A) Two
examples of single-cell [Ca++]i
measurements for cells stimulated with anti-IgE antibody (0.2 µg/ml).
The kinetic timecourse for cells stimulated without PP1 (tracing with a
short timelag interval) is representative of this population of cells,
and the tracing with the longer time interval is representative of
cells stimulated in the presence of 0.8 µM PP1. The cells were
incubated ± 0.8 µM PP1 for 10 min prior to the addition of
stimulus. (B) Kinetics of histamine release in the presence (•) and
absence () of 0.8 µM PP1 (n=2). Cells were incubated
for 10 min with or without PP1 and stimulated with an optimal
concentration of anti-IgE antibody (0.2 µg/ml). At the times shown,
ice-cold PAG containing 10 mM EDTA was added to stop the reaction, the
tubes centrifuged, and the supernatants harvested for analysis of
histamine content. (C) Western blot data for cells incubated ±
PP1 (0.8 µM) for 10 min prior to the addition of anti-IgE at 0.2
µg/ml (n=1). Cells were harvested at the times shown, and
syk was immunoprecipitated and analyzed by Western blot. The top blot
shows the anti-phosphotyrosine blot, and the bottom blot shows the
anti-syk blot [the top blot stripped and reblotted with anti-syk
(4D10)].
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Figure 3. Frequency histograms for the
[Ca++]i timelags for cells
challenged in the presence or absence of PP1. A combination of results
from two preparations of basophils, panel A shows the histogram for
cells stimulated with 0.2 µg/ml anti-IgE antibody, and panel B shows
the results for cells stimulated in the presence of 0.8 µM PP1 (in
both instances, there was a 10-min preincubation period without or with
drug).
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RI involve
tyrosine kinases. Following activation of lyn is the activation of syk
kinase. As will be discussed below, we could not use piceatannol, a
putative, selective, syk kinase inhibitor, but staurosporine and
genistein are now known to inhibit several tyrosine kinases potently.
Each of these drugs has been previously shown to be effective
inhibitors of secretion [36
, 37
] from human
basophils. (Previous studies established the following IC50 values for
inhibition of the histamine release for each of the drugs: PP1, 2 µM;
genistein, 10 µM; staurosporine, 30 nM. The best combination of
forskolin and rolipram rarely causes >50% inhibition of the calcium
signal in basophils, so these drugs were used at concentrations
considered near maximal without causing nonspecific effects. PMA
inhibits the calcium response with an IC50 of 10 ng/ml but causes
histamine release itself, so this value is only relevant to the calcium
response. Bis II does not inhibit histamine release but maximally
inhibits PMA-induced release at 400 nM. For LY294002, there is some
discordance in the inhibition of histamine release—IC50=1–2 µM—and
the [Ca++]i response—IC50=8–10
µM. Historically, the IC50 for inhibition of histamine release by
staurosporine has been increasing steadily for unknown reasons. Our
first studies placed the IC50 at 1 nM [36
]. By the early
1990s, it was found to be between 5 and 10 nM, and by 1996, the IC50
had reached a stable value of 30 nM, the same IC50 found for its
close analog, Go-6936 [38
]. The IC50 for inhibition of
the calcium response by PMA has increased slightly from our original
study [36
], possibly because of changes in how we purify
and handle basophils.)
We found that 100 nM staurosporine inhibited syk kinase
phosphorylation, determined 5 min after the addition of anti-IgE
antibody by 57 ± 15% (unpublished results). The effects of
genistein on IgE-mediated syk phosphorylation haven’t been examined.
Table 1 summarizes the results for the effects of these
drugs on the timelag characteristic. The concentration of the drugs was
chosen to result in only partial inhibition (50%) of the calcium
response to determine the effects on the timelag characteristic.
Although each of the drugs inhibited the average cytosolic calcium
response effectively (net average elevation for the 0–15 min
timeframe), the drugs did not always result in lengthening the timelag.
Even the increases caused by genistein were modest when compared with
the ability of PP1 to increase the timelag. At higher concentrations,
these drugs completely inhibited the
[Ca++]i response;
i.e., there was no timelag to measure. We examined three other classes
of agents known to inhibit the cytosolic calcium response in basophils
[39
], drugs which elevate cAMP levels (a combination of
forskolin and rolipram), protein kinase C (PKC) modulators, and a PI3
kinase inhibitor (Table 1)
. The combination of cAMP-elevating agents
had no effect on the [Ca++]i
timelag but did inhibit the average
[Ca++]i response. The results
with PMA were somewhat more variable, but there remained no
statistically significant increase in the timelag. We also examined the
reasonably selective PKC inhibitor, bis-indolylmaleimide II
[38
], but found no effect of this drug on the timelag.
We have not found a drug that shortened the timelag. Finally, we have
found that LY294002, the selective PI3 kinase inhibitor, inhibits the
IgE-mediated calcium response in human basophils [40
].
However, there was only a modest increase in the timelag that was
evident in both experiments (but not statsitically significant for
n=2).
Previous studies suggested that the IgE-mediated calcium response in
nonreleasing basophils might be nonexistent [6
,
36
]. These results were based on an extrapolation of
results for poorly releasing basophils or relied on data examining the
whole population response under conditions of optimal stimulation. We
have found that the details are more subtle. At the single-cell level,
nonreleasing basophils do, in fact, show a
[Ca++]i response. Figure 4
shows the characteristics of this response under conditions of
optimal stimulation. Figure 4A
shows a typical
[Ca++]i response in
"releasing" basophils and demonstrates the sustained nature of an
oscillating signal. Figure 4B
shows an example of the response of
"releasing" basophils when challenged with an optimal concentration
of anti-IgE antibody with the simultaneous addition of a chelating
concentration of EGTA. This addition allows the continued expression of
the immediate [Ca++]i response as
a result of the release of internal stores of
Ca++ but suppresses the influx phase of the
response. This can be seen in single cells as the steadily decreasing
magnitude of the oscillations until the response is dampened out.
Figure 4C shows the response characteristics of "nonreleasing"
basophils (see Materials and Methods). It is evident that there can be
initial responses with a magnitude similar to releasing cells that
rapidly dampen in a manner similar to that seen in the middle panel.
(It appears unlikely that this is a response to some sort of
contaminant in our anti-IgE preparation. Thus far, there are two types
of calcium response observed in human basophils, those receptors using
heterotrimeric GTP-binding proteins—e.g., fMLP, C5a, PAF—and those
represented by FcRI—with no others currently known. Only activation
through FcRI is known to be inhibited by PP1 currently; this
anti-IgE-antibody response can be inhibited completely with 10 µM
PP1. Therefore, any contaminant would have to utilize a PP1-sensitive
pathway. In addition, the general characteristics of the calcium
response are also consistent with an IgE-mediated response.)
 View larger version (42K): [in a new window] |
Figure 4. Characteristics of the single-cell
[Ca++]i response in basophils
from releasing or nonreleasing donors. Selected examples of the
single-cell response are shown. (A) A representative result for a cell
from a releasing donor. (B) A releasing basophil stimulated in the
presence of 5 mM EGTA. (C) A basophil obtained from a nonreleasing
donor. (D) Average [Ca++]i
response for the two groups of donors, releasers (; n=2)
and nonreleasers (•; n=3). In all cases, the cells were
stimulated with a concentration of anti-IgE antibody considered optimal
for secretion in the microscope chamber, 0.5 µg/ml.
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Figure 5. Frequency histograms for the
[Ca++]i timelag in basophils
obtained from releasing and nonreleasing donors. The top panel shows
the histogram composite for basophil preparations obtained from two
donors with the releasing phenotype. The bottom panel shows the
histogram composite for basophil preparations from three donors
classified as having a nonreleasing basophil phenotype. The mean
timelag values are shown.
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Pharmacological studies indicated that inhibiting the earliest known
step in the FcRI signaling cascade, the activation of lyn kinase,
with PP1 resulted in profound increases in the
[Ca++]i timelag.
Although the concentration of 0.8 µM PP1 could be considered
arbitrary, it was chosen because it resulted in increases in the
timelag similar to those found for the nonreleasers. In this context,
the characteristics of the late calcium response in the presence of PP1
were interesting. At these concentrations of PP1, the net average
increase in calcium for the period of observation was decreased,
because the initial phase of the response was delayed significantly in
onset. The actual elevation, once established, was very similar to the
response without PP1 present. A similar characteristic was observed for
histamine release kinetics. This observation places the results with
PP1 in contrast to the characteristics of the nonreleaser calcium
response (see below).
We did examine other tyrosine kinase inhibitors but were surprised to find that they only poorly increased the duration of timelag. Indeed, staurosporine’s effects were not statistically significant. However, an increase in duration of the timelag did occur in the presence of genistein (p=.024), albeit an increase that was quite modest when compared with the effects of PP1. The lengthening occurring in the presence of staurosporine could have been mitigated, because this drug is also a potent inhibitor of PKC, which has been implicated in down-regulation of immunoreceptors and G-protein-linked, 7-transmembrane-type receptors [43 44 45 46 47 ]. Therefore, we examined the effect of PMA on the timelag and found only modest increases with low concentrations (higher concentrations completely inhibit the calcium response) that were quite variable, which only bordered on statistical significance (p=.057) in these weakly powered studies. (The ratio of timelags in the presence or absence of drug was variable among basophil preparations, although within individual preparations, the distribution of timelags for the two conditions, composed as they were from 30–70 single-cell measurements each, was statistically different for some experiments.) Furthermore, inhibition with potent and reasonably selective PKC inhibitors had no measurable effect on the timelag. Taken together, these data indicate that there is little evidence that PKC plays a role in regulating the duration of the timelag and thus suggest that the variable effect of staurosporine is not easily explained by its potential to inhibit PKC.
The last set of inhibitors we examined were those that elevate cAMP levels or inhibit PI3 kinase. We have shown previously that marked elevations in cAMP cause reasonable decreases in the second phase of the cytosolic calcium response without altering the peak of the first phase of the response [39 ]. Here, we find that not surprisingly, these drugs did not alter duration of the timelag. These results reinforce the previous conclusion that in basophils, the two phases of the calcium response can be regulated independently by cAMP-dependent signaling elements. PI3 kinase has also been implicated in regulating the sustained phase of the [Ca++]i response [48 , 49 ], although we have not seen clear evidence for preferential effects in the human basophil. Wortmannin, another PI3 kinase inhibitor, has been demonstrated to inhibit the [Ca++]i response in basophils [50 ] and LY294002, shown to inhibit secretion and extracellular signal-regulated kinase phosphorylation [51 ]. Studies of wortmannin in RBL cells suggested that there was an increased [Ca++]i timelag [52 ]. With more experiments, the trend to lengthening that we observed might become statistically significant, but compared with PP1 or even genistein, the increase in our studies was quite modest.
Previous studies would have suggested that nonreleasing basophils would
show no calcium response following stimulation with anti-IgE antibody.
However, our own previous studies did not examine previously
characterized, nonreleasing basophils specifically. Results for
nonreleasers could only be extrapolated from linear regressions of
results using cells that had been handled previously for 24 h.
Furthermore, the correlation plot in our former studies was based on
time-average calcium elevations, which as noted below, mask
asynchronous transient elevations (indeed, even a simple linear
regression of the former data would predict a small, nonzero, average
elevation for nonreleasers). In studies by Knol et al.
[6
], nonreleasing basophils were examined directly by
flow cytometry to measure changes in
[Ca++]i. This technique does not
follow the response of a single cell through time so transient
oscillatory elevations that occur asynchronously are not easily
identified. (When observing tens of cells/time interval, a slight haze
of noise above the baseline may be expected to be observed, a
characteristic of the calcium responses from nonreleasing basophils in
the Knol et al. [6
] studies. The problem is
that for these points above the baseline, it is unknown if they are
indeed above their own baseline.) In our current studies, there was an
increase in the duration of the timelag and a transient elevation in
calcium that mimicked the calcium response observed in basophils from
"releaser" subjects stimulated in the presence of EGTA to suppress
the influx phase of the response. In our own studies of the nonreleaser
phenotype, we have not only found markedly reduced expression of syk
kinase but also markedly reduced expression of lyn kinase
[8
]. The donors used in these
[Ca++]i studies were the same
donors we characterized for lyn and syk kinase expression—lyn and syk
kinase were expressed ten–15-fold less in the nonreleasers, and we
found an ninefold lower average
[Ca++]i response. In the PP1
experiments, basophils would experience reduced lyn activity and
therefore reduced syk activity, but the absence of syk in the
nonreleasers might alter the characteristics of the response further.
It is possible that the distinguishing difference between nonreleasers
and releasing cells treated with PP1 is the absence of syk. In this
context, it would be helpful if we could examine the consequences of
syk kinase inhibition. However, the only available putative
syk-selective inhibitor is piceatannol, and we have found that
piceatannol alters basophil responses in unexpected ways for a syk
kinase inhibitor. For example, it inhibits fMLP-induced LTC4 release
with an IC50 lower than required for inhibition of IgE-mediated
histamine release. Furthermore, piceatannol does not inhibit the
[Ca++]i response or the
phosphorylation of Shc in human basophils, an immediate downstream
substrate of syk in a variety of other cell types (unpublished
results).
In summary, these studies showed that weak signaling resulted in prolonged timelags and that the absence of syk and lyn kinase led to the expected increased duration of the timelag in nonreleasing basophils. However, the full character of the calcium response in nonreleasing basophils could not be mimicked by inhibition of lyn kinase using PP1. Surprisingly, other tyrosine kinase inhibitors resulted in little, or at best modest (relative to PP1), increases in the timelag. PKC activation had inconsistent effects, and its inhibition had no effect on the timelag.
Received March 19, 2000; revised October 13, 2000; accepted October 18, 2000.
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, -ß, -
, -, -
in the mast cell line RBL-2H3 J. Immunol. 159,2624-2632[Abstract]This article has been cited by other articles:
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K. Miura, S. Lavens-Phillips, and D. W. MacGlashan Jr. Localizing a Control Region in the Pathway to Leukotriene C4 Secretion Following Stimulation of Human Basophils with Anti-IgE Antibody J. Immunol., December 15, 2001; 167(12): 7027 - 7037. [Abstract] [Full Text] [PDF]
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