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RI
up-regulation by IgE in human basophils
Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland
Correspondence: Donald MacGlashan, Jr., Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: dmacglas{at}welch.jhu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RI
by immunoglobulin E (IgE) for human
basophils. Basophils were cultured with and without IgE antibody (PS
myeloma IgE or anti-gp120-specific IgE) for 1 week and challenged with
anti-IgE, anti-FcRI, or antigen for histamine and IL-4 secretion.
There were no statistically significant changes in their response to
anti-IgE or anti-receptor antibodies, as compared with controls
incubated for the same period, whereas receptor expression increased an
average of 4-fold. There was increased responsiveness to antigenic
challenge, most notably at suboptimal concentrations of antigen (gp120
peptide-ovalbumin conjugate). For a 6-fold difference in cell surface
density of gp120-specific IgE, there was a 2.2-fold change in antigen
potency or 3-fold increases in histamine release at lower antigen
concentrations. Similar results were found for secretion of IL-4.
Basophil sensitivity, which is a measure of the density of
antigen-specific IgE required for 50% of maximal secretion, was used
to determine whether up-regulation of FcRI was coordinated with
up-regulation of other components of the IgE-signaling pathway. The
results indicated up-regulation of FcRI is not always accompanied by
changes that allow sensitivity to be maintained. These results indicate
that functional up-regulation does occur but that its magnitude may be
modulated because not all components of the signaling pathway are
up-regulated in a balanced manner.
Key Words: FcRI • immunoglobulin E • basophils
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RI rather than through
some other IgE-binding protein [5
]. The mechanisms of
the up- or down-regulation mediated by IgE remain unknown and are under
investigation. However, it is apparent from studies in mice that
up-regulation of FcRI is accompanied by a better response to
IgE-dependent stimuli [6
]. For example, up-regulation of
FcRI on bone marrow-derived mast cells during 1 week of culture with
IgE led to marked increases in IL-4 secretion and modest changes in
serotonin release. Based on a variety of studies over the last several
decades, it is not surprising that enhanced functionality accompanies
the increased expression of FcRI, especially when the starting cell
surface density is quite low, as it is in relatively young bone marrow
mast cells from mice. Similar experiments are not as straightforward
for the human basophil. To culture basophils for an extended period,
interleukin 3 (IL-3) must be included in the culture medium, and IL-3
itself is a powerful means to alter basophil function. In addition, the
starting density of receptors on basophils is relatively high, so that
the degree of receptor up-regulation is often quite modest
[3
]. Therefore, recently published studies have not
focused on the functional changes that might accompany the
up-regulation of FcRI in human basophils. The current studies begin
to explore the characteristics of any changes that might occur.
There is an additional reason for exploring the nature of any changes
that are related to secretion. It is now recognized that there may be
components of the IgE-mediated signaling pathway that are
rate-limiting. Examples might be the src-family kinase, lyn
[9
, 10
], or the ß subunit of FcRI
itself [11
], which has been demonstrated to be
unnecessary for cell surface expression of FcRI
[12
13
14
]. Although there is no direct proof that these
two particular components are required for signaling in human
basophils, all available evidence indicates that they are. It is
possible that there are other required components that may be
rate-limiting. For the human histamine-containing FcRI-expressing
cells studied to date, freshly isolated or developed in culture,
up-regulation has only been demonstrated for FcRI, not other
components of the receptor or any other accessory molecules related to
IgE-mediated signaling. Rather than attempt to evaluate the
up-regulation of all potential components, we have asked whether the
characteristics of the functional response can provide some insight
into whether up-regulation of FcRI is balanced by concordant
up-regulation of other components of IgE-mediated signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
Anti-antibody, 22E7, was a gift from Dr. Jarema Kochan at
Hoffman LaRoche (Little Falls, NJ). Polyclonal goat anti-human IgE was
prepared as described previously [16
]; the antibody used
for these studies represented the IgG fraction of goat serum prepared
by DE-52 chromatography. Purified IgE-PS myeloma was a gift from Dr. T.
Ishizaka [17
]. Anti-gp120 chimeric IgE and gp120
peptide-ovalbumin conjugate (gp120-OVA) were gifts from Dr. Frances M.
Davis of Tanox Biosystems (Houston, TX) and were prepared by methods
previously described [18
]. IL-3 was obtained from
Biosource (Camarillo, CA).
Cell preparation
We used two types of basophil preparations. Many of the studies
used cells obtained from leukapheresis and were prepared as previously
described [19
]. Basophil purities in these preparations
ranged from 15 to 95% with a median of 33. In some experiments, cells
were isolated by the double-Percoll method used for fresh blood
[20
]. The blood was diluted with EDTA-saline,
centrifuged at 500g for 15 min to obtain a buffy coat. The
buffy-coat cells were diluted in saline and layered onto a 2-step
Percoll gradient, 1.072 g/ml/1.082 g/ml as described previously
[21
]. After centrifugation at 700g for 20
min, the interface between the 1.072 Percoll/plasma upper layer and the
1.082 lower Percoll layer was harvested and washed as above (basophil
purities of 8–45%). We noted in the text which cell preparations we
used.
Cell culture
To modulate FcRI expression, enriched basophil
preparations were cultured in Iscove’s modified Dulbecco’s media
(IMDM) (Life Technologies) containing 2% FCS, 40 µg/ml gentamicin,
and 10 ng/ml of IL-3 with or without IgE. The total cell density was
2 x 106/ml and culturing was done in 96-, 24-, or
6-well tissue culture-treated plates (Costar, Cornell, NY). For
secretion of IL-4 and IL-13, basophils were cultured in C-IMDM (IMDM
conditioned with 5% heat-inactivated [56°C for 20 min.] FCS,
nonessential amino acids [Life Technologies], and 10 µg/ml
gentamicin), as reported elsewhere [20
]. Cytokine
protein measurements and percent histamine released in the same culture
supernatants were assessed by enzyme-linked immunosorbent assays and
automated fluorimetry, respectively [20
].
Sensitization methods
Previous studies established that 3–10 µg/ml of IgE would
optimally up-regulate expression of FcRI in 1-week cultures
[3
]. For many of the experiments, anti-gp120 hIgE was
used at 5–10 µg/ml in these cultures. For the experiments shown in
Figures 3, 5, and 6, anti-gp120 IgE was mixed with PS myeloma IgE so
that the total concentration of IgE was maintained at 10 µg/ml. The
concentrations of gp120-IgE were 10 µg/ml (with no PS myeloma), 3.3,
1.1, 0.34, and 0.11 µg/ml (the last four with PS myeloma IgE at 6.7,
8.9, 9.66, and 9.89 µg/ml, respectively). Two groups of cells were
cultured for 1 week in IMDM medium supplemented with nonessential amino
acids, 2% FCS, 40 µg/ml gentamicin, and L-glutamine: one group of
five conditions, where IgE was included at the concentrations noted
above for the entire week, and a second group of five conditions that
were sensitized with the above concentrations of gp120-IgE 1 h
before harvesting the cells (at the end of the 1-week culture).
IgE sensitivity curves for cytokine secretion (Fig. 3) were assessed using basophils passively sensitized with varying levels of gp120-IgE and then challenged with specific antigen. For most, but not all, of the experiments, passive sensitization was performed by first removing receptor-bound IgE using a lactic acid protocol modified from that previously reported. Briefly, basophil-enriched cell suspensions prepared by double-Percoll density centrifugation were washed 1x with 1 ml of ice-cold 0.9% NaCl in 1.5-ml tubes and centrifuged at 150g for 5 min. Cell pellets were resuspended in 0.25 ml ice-cold lactic acid buffer for 30 sec before adding 1 ml sensitization buffer with IgE. After 10 min on ice, the cells were centrifuged, the supernatants aspirated, and the pellets resuspended once again in sensitization buffer. Cells were aliquoted in 100-µl volumes, to which varying amounts of gp120-IgE mixed with PS myeloma IgE were added as described above. After incubating 30 min at 37°C, the cells were washed 3x with PAG.
Flow cytometry
A flow cytometric technique incorporating light scatter
characteristics was used to quantify cell surface IgE and FcRI
chain expression on basophils as described [22
]. Cell
surface IgE that was specific for gp120 peptide was detected using a
monoclonal antibody (AB19-4, Tanox). Using enriched basophil
preparations at purities >25%, this antibody can detect <1,000
molecules of gp120-specific IgE. This antibody does not detect other
cell-bound IgE. Cells not sensitized with gp120-specific IgE showed
flow cytometric distributions equal to nonspecific IgG1 labeling of
cells sensitized with gp120-specific IgE. Cell surface expression of
FcRI chain was detected using a mouse IgG1 anti-human FcRI
chain monoclonal antibody (22E7, provided J. Kochan, Roche, Mont Clair,
NJ [23
]) and was compared with labeling with an
identical concentration of irrelevant mouse IgG1 (Coulter, Hialeah,
FL). The 22E7 antibody has been shown to recognize an epitope that is
unaffected by FcRI occupancy [23
]. Aliquots of
cells were labeled in phosphate-buffered saline containing 0.2% HSA
with 1 mg/ml human IgG to minimize nonspecific binding to Fc
R
[22
]. Each of the monoclonals were used at
concentrations predetermined to be optimal for labeling. Binding of
monoclonals was detected using saturating concentrations of
R-phycoerythrin-conjugated polyclonal goat anti-mouse IgG (Tago,
Burlingame, CA). An EPICS Profile flow cytometer (Coulter) was used to
analyze fluorescent signals after excitation at 488 nm. "Bitmap"
gates, intermediate between the forward and side scatter
characteristics of lymphocytes and monocytes, were used to select for a
population of cells that were predominantly basophils. Because the
cells were already enriched in basophils, these bitmaps can select a
population of cells that is generally >80% basophils, with the
primary contaminants being lymphocytes. Data are expressed as the mean
fluorescence in labeled cells minus the mean fluorescence of IgG1
controls. Day-to-day variability in the sensitivity of the flow
cytometer was corrected by noting or adjusting the photomultiplier tube
voltage to generate the same signal for a set of standard calibration
beads (Immunochek, Coulter).
In previous studies [3
], the flow cytometric
measurements were calibrated examining the fluorescence staining of six
donors’ basophils that spanned a moderate range of staining
intensities (7–120 fluorescent units or 8,000–140,000 FcRI per
basophil) and simultaneously assessing receptor or IgE density by the
acetate elution method described above. 22E7 staining (ordinate)
compared with total FcRI density by acetate elution (after
sensitizing with PS myeloma IgE as described above) was linear with a
slope of 0.00084 (i.e., a fluorescence measurement of 100 represents
120,000 receptors) with r = 0.963. gp120-specific
IgE density as detected with AB19-4 anti-id antibody was previously
calibrated by sensitizing RBL-SX38 cells (expressing a transfected
human FceRI) with gp120-IgE, stripping and measuring total IgE in a
RIST [24
]. When combined with cell counts, these
measurements established the cell surface gp120-IgE density with which
the fluorescent measurements were compared.
Statistics
Some statistical comparisons were made with a Student’s t-test,
whereas others where made with a nonparametric Wilcoxon’s signed rank
statistic. If error bars are shown, they represent the standard error
of the mean unless otherwise indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RI antibodies to determine if the dose
response curves to these stimuli differed in cells that been exposed to
IgE for 1 week versus those that had not. For these experiments,
partially enriched basophils were cultured for 1 week in the presence
or absence of PS myeloma IgE at 5 µg/ml in IMDM media containing 10
ng/ml of IL-3. Expression of FcRI was determined on day 0 and day
7 by flow cytometry using 22E7 as the detection antibody. On day 7,
cells were harvested and challenged with several concentrations of 22E7
or polyclonal anti-IgE antibody. As can be seen in Figure 1
, there was no statistically different response between the two
groups of cells and any concentration of antibody. Receptor expression
increased an average of 4 ± 1-fold. Based on studies where flow
cytometric measurements of 22E7 binding were calibrated against
receptor density measurements by the acetate strip method, these cells
started with 15,000 receptors on day 0 and ended with 53,000
receptors on day 7. The cells incubated without IgE expressed 15,000
receptors on day 7 (no statistical change from day 0).
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33,000 gp120-IgE molecules per cell, whereas those
incubated for 1 week with gp120-IgE were found to be loaded with
188,000 gp120-IgE molecules per cell.
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RI and
stimulation with antigen, IL-4 secretion was marked [6
].
Therefore, we examined two facets of the basophil response in context
with histamine release: IL-4 and IL-13 secretion. In previous studies,
we described a technique to assess the sensitivity of human basophils,
with sensitivity defined as the number of antigen-specific IgE
molecules required to attain 50% of the cell’s maximum response
[15
] (see discussion for the background leading to this
definition). If IL-4 or IL-13 secretion were differentially sensitive
to the strength of the stimulus, the sensitivity curves for histamine,
IL-4, and IL-13 release would be different, and, if different (in the
correct direction), then up-regulation of receptor expression by IgE
could lead to results similar to those observed in murine mast cells
for IL-4 secretion. Therefore, we first examined the sensitivity curves
for the secretion of histamine, IL-4, and IL-13. For these experiments,
it was not necessary to know the absolute number of IgE molecules
loaded into unoccupied receptors, only the relative response of each
secreted mediator from basophils sensitized with different amounts of
IgE. Figure 3
shows these results. Serial dilutions of anti-gp120 IgE were used
to sensitize basophils, which were then washed and challenged in IMDM
medium containing 5% FCS and gentamicin (see Materials and Methods).
Supernatants were harvested at 4 and 24 h. The figure shows that
the sensitivity curves were similar for the release of the IL-4 and
histamine (data for IL-13 was similar but not shown). Similar results
were found for cells not treated with lactic acid (data not shown). The
similarity in sensitivity for the three mediators implies that
up-regulation of FcRI by IgE should not lead to results seen in
murine mast cells (see Discussion). To verify this conclusion, we
examined a single preparation of basophils for IL-4 secretion after 7
days of culture with and without IgE antibody. As observed for
histamine release, there was a 2-fold difference in antigen potency for
cells up-regulated with or without gp120-specific IgE for 1 week (data
not shown).
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RI in human basophils, and a
variety of studies indicate that there may be elements of the signaling
pathway that may be rate-limiting and, therefore, also require
up-regulation for secretion to be enhanced. We have developed an
alternative method for examining cell responsiveness that should also
address the issue concerning rate-limiting accessory molecules.
Figure 4
illustrates the logic behind the procedure. Basophil sensitivity
is defined as the density of antigen-specific IgE molecules required
for 50% of the cells maximum IgE-mediated response. In the complete
method (unlike the experiments shown in Figure 3
), the amount of
antigen-specific IgE that is present on a sensitized cell is measured.
Thus, for each sensitization condition, two parameters are assessed:
the density of antigen-specific IgE and the amount of histamine release
after challenge with an optimal concentration of antigen. The
sensitivity curve labeled ’1’ in Figure 4
is based on both the number
of antigen-specific IgE molecules per cell, which are plotted along the
abscissa and the overall strength of the signal reflected in the
histamine release, is plotted on the ordinate. In this depiction, the
signal strength is essentially dependent on all those factors that are
not FcRI/IgE itself, represented by the filled circles. After
up-regulation of FcRI, Figure 4
depicts no change in the
non-FcRI accessory factors, therefore, the signal generated is
the same as in the original cells. However, because cell-surface
antigen-specific IgE is measured and is increased due to up-regulation
of FcRI, the point that is plotted is shifted to the right.
Therefore, the entire curve is shifted to the right. On a per
FcRI/IgE basis, the basophil appears less sensitive. The figure
shows this for a case where FcRI density increases by 4-fold
without changes in the accessory molecules. On the other hand, if
accessory molecules also increase in proportion to FcRI, then the
relative sensitivities should appear identical. If accessory molecules
increased more than FcRI, then the cells would appear more
sensitive. Finally, if FcRI is the rate-limiting species, and all
other participating species are in excess, then increasing FcRI
without increasing the accessory molecules also results in the two
curves being similar or identical.
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RI would be occupied by the IgE used for
up-regulation). However, for each dilution of gp120-IgE, the same
degree of total FcRI up-regulation needed to be maintained. In
this way, the hypothetical increase in accessory molecules would be the
same for each condition with only the density of gp120-specific IgE
varying among conditions. Therefore, the IgE concentration was held
constant at 10 µg/ml by diluting the gp120-IgE with medium containing
10 µg/ml of PS myeloma. The assumption was that gp120-IgE antibody
and PS myeloma IgE antibody induced the same degree of receptor
up-regulation. This assumption was tested in pilot experiments by
comparing the potency of these two IgE antibodies for up-regulating in
1-week cultures. In data not shown from five experiments, the relative
potency of the two IgE antibodies was found to be statistically
identical. This assumption was also born out by the equivalent amount
of FcRI up-regulation observed for each of the different
conditions (as detected by 22E7 binding). Partially enriched basophil
preparations (these experiments required 10 million basophils to
compensate for losses during the 1-week culture) were divided on day 0
into a group incubated without IgE and those incubated with IgE. Those
cells cultured without IgE for 1 week were sensitized 1 h before
harvesting the cells (see Materials and Methods). The cells were
analyzed for histamine release in response to an optimal concentration
of gp120-OVA (50 ng/ml) or subjected to flow cytometry using either
22E7 or anti-idiotype antibody AB19-4 (anti-idiotype antibody for the
gp120-specific IgE).
Figure 5
shows the results from one of these experiments. The total
FcRI expression differed by 4-fold between the two groups of
cells (those treated for 1 week with IgE vs. those without IgE and
sensitized just before harvesting). Likewise, it can be seen that the
relative sensitivity was shifted by 4-fold. This result parallels
that shown schematically in Figure 4 . However, we found that results
varied among preparations of basophils. Because the amount of
up-regulation differed for each preparation, it was more instructive to
plot individual experiments as shown in Figure 6
. On the abscissa is the fold up-regulation of FcRI, on the
ordinate in the fold difference in the two sensitivity curves. A
priori, there is no reason to preclude sensitivity being greater after
up-regulation, which would plot a point on the negative ordinate, but
this was not observed. The dotted line at 45° represents the location
for points where, under the model of some accessory elements being
rate-limiting, one would find up-regulation of FcRI in the
absence of up-regulation of any other accessory element. Data points
that fall on the horizontal line would be less interpretable. As noted
above, this latter situation could result from coordinated
up-regulation of all components where accessory species are
rate-limiting or from uncoordinated upregulation when FcRI is the
rate-limiting species. From this perspective, in three experiments,
functional up-regulation of all relevant FceRI signaling molecules
appeared uncoordinated, either lying on the 45° line or between the
two dotted lines. In two experiments, the sensitivity curves were
identical and, thus, the points lay on the horizontal line. For one of
these experiments (marked with an asterisk in Figure 6
), a day 0
sensitivity curve was also generated; for this one experiment the
sensitivity at day 7 (in cells not treated with IgE) was 2-fold
lower than the sensitivity of day 0, indicating a modest loss of
responsiveness in cells cultured for 7 days despite the presence of
IL-3.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in function are generally better appreciated by using antigens
to aggregate specific IgE. The experiments shown in Figure 2
continue
to support this view. The range of antigen-specific IgE that was
examined was more appropriate to search for up-regulation and, because
the stimulus was an antigen, broadening of the dose response curve was
expected. However, there was a 6-fold difference in the density of
gp120-specific IgE between the two conditions and only a 2.2-fold
increase in potency. For some of the lower concentrations of antigen,
there was only a 3-fold increase in absolute histamine release between
the two levels of receptor expression. One context for evaluating the
significance of these changes can be found in the murine studies where
up-regulation of FcRI caused marked changes in IL-4 secretion. In
these experiments, the fold change in secretion was certainly much
greater than the fold change in receptor expression. We could find no
evidence that release of cytokines was any more or less sensitive to
stimulation than degranulation and that up-regulation of this response
was, therefore, similar to histamine release. The studies in mice had
the advantage that starting cell-surface densities of FcRI were very
low. However, the implication of the murine studies is that if a
sensitivity curve were generated for mouse mast cells or basophils like
the one shown in Figure 3
, the curve for IL-4 secretion would be
different than the one for serotonin release, possibly 3–10-fold
shifted to the right of the curve for serotonin release
[6
]. Our results for human basophils indicate little
difference in the curves, or possibly an IL-4 sensitivity curve that is
shifted leftward of the histamine release curve, a result opposite of
that expected for murine cells. Based on the sensitivity curves shown
in Figure 3 , we would have predicted that up-regulation of IL-4 release
after incubation for 7 days with IgE would be no different than that of
histamine release. This was verified experimentally. One caveat to the
studies shown in Figure 3
is that the IL-4 sensitivity curve shows an
optimum in cells that were sensitized with 3 µg/ml of gp120-IgE. This
characteristic, where too much signaling causes a decreased IL-4
response, has been observed for stimulation with anti-IgE antibody
[19
, 28
]. Our working hypothesis is that
the observation reflects the increasing rates of desensitization at
higher levels of signaling so that a short-lived secretory event like
degranulation is less effected by an increased rate of desensitization
than is a long-lived secretory event like IL-4 release.
Another context in which to view the significance of the difference in antigen dose response curves in Figure 2 is found in the characteristics of basophil sensitivity. The control sensitivity curve shown in Figure 5 is representative of many other sensitivity curves demonstrated in previous studies. It can be seen that at low levels of sensitization, there are disproportionate changes in basophil function. For example, histamine release was 4% when gp120-specific IgE density was 2 flow units and 36% for 8 flow units. A similar disproportionality was observed in the recent studies of recovery from MAb E25 treatment in atopic patients. A critical point of these studies was the observation that a 3-fold change in receptor expression, at the low end of the receptor expression, was accompanied by a 5-fold increase in function [4 ]. The critical point of these studies is that disproportional changes in function versus cell-surface IgE occur most obviously at low densities of IgE, whereas the changes in function for the current studies occur at higher densities of cell-surface IgE.
A third context for evaluating the differences in antigen-induced
response comes from the sensitivity studies shown in Figures 4
5
6
.
Ideally, it might seem that the best definition of sensitivity would be
the number of cell-surface aggregates required to elicit a fixed amount
of mediator release. However, it was shown both in human basophils and
rat basophilic leukemia cells that not all aggregates can be considered
equal with respect to the signals they initiate
[29
30
31
]. Therefore, such a definition of sensitivity
would also have to specify the precise nature of the aggregate: its
size, complexity (extent of branched aggregation), and duration. This
is, of course, not practical. However, for comparisons among basophil
donors, a reasonable approach is to define sensitivity on the basis of
the number of antigen-specific IgE molecules per cell, provided that
the same IgE were used on each basophil preparation examined and the
same antigen used to stimulate the cells. Under these conditions, one
could reasonably assume that the characteristics of the aggregates
would be similar. This was done for previous studies of basophil
sensitivity. A further consideration for defining sensitivity is to
decide whether the response is expressed as a function of the absolute
level of secretion (e.g., percent histamine release, fg of IL-4
secreted per basophil, etc.) or relative to the maximum release
obtainable with any IgE-dependent stimulus. We previously showed that
maximum release and number of molecules of IgE required for 50% of
maximum release were independent parameters of cell function
[15
]. One parameter could change without the other
changing. For the current studies, the preconditions for measuring
sensitivity could have been relaxed somewhat because no comparisons
were made between basophil preparations; only differences in
sensitivity within preparations was determined. Furthermore, because
the cells within each basophil preparation were cultured under the same
conditions, with the exception of the presence or absence of IgE, we
found no differences in the maximum release between the two conditions.
In data generated for previous studies, we also noted that the
qualitative characterisitics of the sensitivity curves do not change if
suboptimal, rather than optimal, concentrations of antigen are used to
stimulate the cells after sensitization. The experiments shown in
Figure 5 , therefore, represent a well-controlled assessment of basophil
sensitivity. The uncoordinated up-regulation of all the signaling
components suggested by the results shown in Figures 5
and 6
should
provide an additional context for evaluating the data in Figure 2
. In
other words, functional up-regulation within a given preparation of
basophils may depend on more than up-regulation of FcRI alone.
Variability among experiments might be explained by the varied degree
of uncoordination (Fig. 6)
.
Growing evidence shows that not all components of the IgE-mediated
signaling cascade are present in constant stoichiometric quantities.
Beginning with the receptor itself, FcRIß is not necessary for
expression of FcRI. We will be reporting studies that indicate
that /ß stoichiometry is not constant across a range of FcRI
densities [32
]. Because FcRIß is an amplifier of
the earliest stage of the signaling cascade [11
],
uncoordinated regulation of these two components alone could account
for a large proportion of the variability in IgE-mediated function we
observe. Some indication that such uncoordination of FcRIß
expression can be found in the studies of human liver-derived mast
cells [33
]. In these studies, culture with IL-4
up-regulated the mRNA for FcRI but not FcRIß or FcRI.
Although levels of mRNA don’t always readily translate to protein
expression, these studies are suggestive.
Studies in RBL cells indicate that lyn kinase is a rate-limiting component of the reaction [9 , 10 ]. Although these results are somewhat controversial and not necessarily applicable to human basophils, they raise the issue that increased function might only follow coordinated changes of any component in the signaling cascade that is otherwise rate-limiting. The studies shown in Figures 5 and 6 indicate that coordination of up-regulation is an issue, and, at a minimum, indicate by another means the possible variability in the results for different preparations. Taken at face value, these results indicate a new point of regulation in the responsiveness of these cells to stimulation. The factors regulating the coordinated up-regulation would be a future target of study. An important caveat for these in vitro studies is that the basophils are incubated for 1 week. Although IL-3 appears to maintain functionality, cells are lost at rates that vary among preparations. Either the average rate of loss reflects the natural life span of basophils in vivo, or we have yet to provide the correct mix of cytokines to sustain normal functionality in culture. Differential sensitivity to an inadequate cytokine environment could explain the variable results shown in Figure 6 . However, even if this were true, the results continue to indicate that up-regulation requires co-factors that determine the degree of balanced expression of all necessary components of the reaction.
In summary, IgE induces up-regulation of FcRI with a resulting
increase in basophil responsiveness to antigenic challenge. However,
the resulting up-regulation of function may be regulated by other
factors, differing among preparations, because up-regulation of
FcRI is not always coordinated with other yet-to-be-identified
components of the IgE-mediated signaling cascade.
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ACKNOWLEDGEMENTS |
|---|
Received January 1, 2000; revised March 23, 2000; accepted April 26, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RI surface densities and mediator release by anti-IgE infusions is reversible in vitro and in vivo J. Immunol. 162,5623-5630
RI and FcRI levels in human basophils J. Allergy Clin. Immunol. 105,S172
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D. MacGlashan Jr. and N. Vilarino Nonspecific Desensitization, Functional Memory, and the Characteristics of SHIP Phosphorylation following IgE-Mediated Stimulation of Human Basophils J. Immunol., July 15, 2006; 177(2): 1040 - 1051. [Abstract] [Full Text] [PDF]
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D. MacGlashan Jr. Two Regions of Down-Regulation in the IgE-Mediated Signaling Pathway in Human Basophils J. Immunol., May 15, 2003; 170(10): 4914 - 4925. [Abstract] [Full Text] [PDF]
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) or without (•) 5 µg/ml PS myeloma in media
containing 10 ng/ml of IL-3. After 7 days, the cells were harvested and
challenged with three concentrations of anti-FceRIa antibody, 22E7
(panel A), or one concentration of anti-IgE antibody (panel
B, - and + indicate the absence or presence of IgE in
the culture, respectively). Supernatants were analyzed for histamine
content. Not indicated, receptor expression increased by 4 ±
1-fold in the cells cultured with IgE.
