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Receptor I Expression and IgE-Dependent Release of Histamine and Lipid Mediators from Human Umbilical Cord Blood-Derived Mast Cells: Synergistic Effect of IL-4 and IgE on Human Mast Cell Fc Receptor I Expression and Mediator Release1
Departments of
*
Pathology and
Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215;
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; and
§
Department of Immunology, Juntendo University, School of Medicine, Tokyo, Japan
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI surface
expression in differentiated human mast cells derived in vitro from
umbilical cord blood mononuclear cells. We found that IgE (at 5
µg/ml) much more strikingly enhanced surface expression of FcRI
than did IL-4 (at 0.1–100 ng/ml); similar results were also obtained
with differentiated mouse mast cells. However, IL-4 acted
synergistically with IgE to enhance FcRI expression in these
umbilical cord blood-derived human mast cells, as well as in mouse
peritoneal mast cells derived from IL-4-/- or
IL-4+/+ mice. We also found that: 1) IgE-dependent
enhancement of FcRI expression was associated with a significantly
enhanced ability of these human mast cells to secrete histamine,
PGD2, and leukotriene C4 upon subsequent
passive sensitization with IgE and challenge with anti-IgE; 2)
preincubation with IL-4 enhanced IgE-dependent mediator secretion in
these cells even in the absence of significant effects on FcRI
surface expression; 3) when used together with IgE, IL-4 enhanced
IgE-dependent mediator secretion in human mast cells to levels greater
than those observed in cells that had been preincubated with IgE alone;
and 4) batches of human mast cells generated in vitro from umbilical
cord blood cells derived from different donors exhibited differences in
the magnitude and pattern of histamine and lipid mediator release in
response to anti-IgE challenge, both under baseline conditions and
after preincubation with IgE and/or IL-4. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) expressed on the surface of cells (8, 9). Moreover, it has long been known that patients with parasite
infections or allergic diseases typically have elevated concentrations
of IgE (1, 7, 10), and that atopic subjects with high levels of serum
IgE also exhibit increased expression of FcRI on the surface of
blood basophils (11, 12). Yet, until recently, relatively little
attention had been given to the possibility that IgE itself might
regulate the ability of mast cells (or other FcRI+
effector cells) to bind IgE, or that this might have important
implications for mast cell function.
However, it is now clear that IgE can enhance the IgE-binding ability
of bone marrow-derived mouse mast cells in vitro (13, 14) and mouse
peritoneal mast cells in vitro or in vivo (13), and that this increased
IgE-binding ability reflects enhanced cell surface expression of
FcRI (13). The administration of IgE also can increase the
expression of FcRI by rat mast cells in vivo (15). Moreover, such
IgE-dependent up-regulation of FcRI expression can significantly
increase the ability of mouse mast cells to release preformed mediators
or cytokines in response to challenge with specific Ag (13).
IgE-dependent enhancement of mouse mast cell FcRI expression had a
particularly striking effect on the ability of cells to secrete IL-4
(13), a cytokine that can promote the production of IgE (16, 17).
While the relevance of these observations to the human system was
not initially clear, we recently reported that IgE can enhance the
IgE-binding ability of umbilical cord blood-derived cultured human mast
cells, and that such IgE-dependent enhancement of the IgE-binding
ability of cells enhances the ability of these anti-IgE-induced
mast cells to secrete histamine, and the chemokine
macrophage-inflammatory protein-1
(MIP-1),6 in response to
challenge with anti-IgE (18). Notably, Kimata et al. (19) have
reported recently that MIP-1 can enhance IgE production by human B
cells in vitro.
In addition to IgE, IL-4 can also enhance FcRI expression in certain
populations of human mast cells. Toru et al. (20) reported that IL-4
can up-regulate FcRI expression and IgE binding in human mast cells
derived in vitro from CD34+ human umbilical cord blood
mononuclear cells maintained in stem cell factor (SCF) and IL-6 without
PGE2, and Pawankar et al. (21) found that IL-4 can also
enhance surface expression of FcRI on human nasal mast cells in
vitro. While neither of those studies compared the effects of IL-4
versus IgE on human mast cell FcRI expression, H.-Z. Xia et al. (22)
recently did perform such a comparison, using fetal liver-derived human
mast cells that had been generated in SCF-containing media in vitro.
These fetal liver-derived human mast cells, which developed over a
period of 4 wk in vitro in the absence of exogenous rhIL-4, expressed
almost no detectable FcRI on the cell surface (22). In these mast
cell populations, Xia et al. found that IL-4 was substantially more
potent than IgE alone in inducing the expression of FcRI, but that
IL-4 and IgE had a synergistic effect. Moreover, the ability of IL-4 to
enhance FcRI expression progressively diminished during the first 2
wk of culture, indicating that the cytokine is most effective in
inducing FcRI expression in this system if it is present during the
earliest stages of mast cell differentiation (22).
In the present study, we analyzed the effects of IgE versus IL-4 on
FcRI expression in a different population of human mast cells:
differentiated mast cells generated in SCF-containing medium from
umbilical cord blood mononuclear cells. We find that, in these human
mast cells, IgE is substantially more potent than IL-4 in enhancing
FcRI expression, and that IgE-dependent enhancement of FcRI
expression permits the cells to secrete increased amounts of lipid
mediators, in addition to histamine, in response to challenge with
anti-IgE. However, when added with IgE, IL-4 can enhance mast cell
surface expression of FcRI over that observed in cells that had been
preincubated with IgE alone; IL-4 preincubation can also enhance mast
cell mediator secretion in response to anti-IgE challange even
without producing significant enhancement of FcRI surface
expression. Some of these results have been reported in abstract form
(23).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro derived human mast cells were obtained essentially as
described in Saito et al. (24). Heparinized cord blood (Advanced
Biotechnologies, Columbia, MD, or Brigham and Women’s Hospital,
Boston, MA) was overlaid on Histopaque 1077 (Sigma, St. Louis, MO) and
centrifuged at room temperature for 30 min at 400 x g.
Mononuclear cells at the interface between plasma and Histopaque 1077
were collected, washed, and then maintained in culture medium,
consisting of Iscove’s modified Dulbecco’s medium (IMDM; Sigma)
supplemented with 10% FCS (HyClone, Logan, UT); 80 ng/ml human
rSCF164 (rhSCF; Amgen, Thousand Oaks, CA) (25); 50 ng/ml
human rIL-6 (rhIL-6; Amgen); 1 µM PGE2 (Cayman Chemical,
Ann Arbor, MI); and from Life Technologies (Gaithersburg, MD), 10 mM
HEPES, 2 mM L-glutamine, antibiotics (100 U penicillin/ml,
100 µg streptomycin/ml, and 10 µg gentamicin/ml), 1 x MEM
vitamin solution (catalogue 11120), 1x MEM amino acids (without
L-glutamine, catalogue 11130), 1 mM sodium pyruvate, and 50
µM 2-ME. Half of the culture medium was changed weekly, and cells
were cultured for at least 9 wk. The purity of the mast cells at the
time of individual experiments was 86 to >99% (as specified in the
text), as determined using Kimura’s stain (26) and/or tryptase
immunostaining (24); the two methods usually gave essentially identical
results. In some experiments, mast cells were incubated (unless
specified otherwise, in the usual culture medium) for up to 8 days with
or without purified human myeloma IgE (Biodesign International,
Kennebunk, ME, at 0.05 or 5 µg/ml microcentrifuged at 
14,000
x g for 20 min at 20°C to remove possible aggregates),
and/or rhIL-4 (Genzyme, Cambridge, MA), before analyses of mast cell
surface FcRI expression and/or mediator release (see below). After
centrifugation, IgE was removed from the top 20% of the solution for
incubation with mast cells; the IgE concentrations of solutions before
or after centrifugation were 104.6 ± 2.6% or 101.2 ± 2.6%
of the supplier’s reported values, and the difference between these
values was not statistically significant. Mast cell numbers and
viability (which was always >90% according to trypan blue staining)
were assessed immediately before analysis of mast cell FcRI surface
expression or mediator release (i.e., after culture and/or
preincubation for up to 8 days ± IgE or IL-4).
Flow cytometry of umbilical cord blood-derived human mast cells
Cells were washed once in DMEM (Life Technologies) supplemented
with 3% FCS (Sigma), then preincubated with human IgG (Sigma) at 10
µg/ml for 15 min, and then incubated at 4°C with purified human
myeloma IgE (Biodesign International) at 10 µg/ml for 50 min; pilot
studies showed that these conditions resulted in 96% of the level
of binding of IgE to mast cells as was obtained with cells that had
been incubated with IgE at 50 µg/ml for 150 min at 4°C. After
washing once in DMEM supplemented with 3% FCS (Sigma), cells were
stained with FITC goat anti-human IgE Ab (Biosource International,
Camarillo, CA; at 9 µg/ml) for 25 min at 4°C. To analyze directly
levels of mast cell surface FcRI expression, cells were preincubated
with human IgG for 15 min (as above) and then incubated for 1 h at
4°C with 5 µg/ml of the mouse IgG2b anti-human FcRI
-chain mAb, CRA-1, which can bind to the FcRI -chain whether
or not it is occupied by IgE (21, 27). After washing, cells were
stained with FITC goat F(ab')2 against mouse IgG (Jackson
ImmunoResearch, West Grove, PA) at 7.5 µg/ml for 30 min. An
isotype-matched mouse IgG2b mAb with irrelevant specificity (MOPC195;
Organon Teknika, Durham, NC) was used as negative control instead of
CRA-1. Stained cells were analyzed using a FACSCalibur (Becton
Dickinson, San Jose, CA). Ten thousand events in each sample were
analyzed, and at least five thousand mast cells were studied to
calculate the median value of fluorescence intensity.
An instrument setting of the flow cytometer that was appropriate for analysis of human mast cells was determined in the first experiment and was used for all such analyses throughout this study. In addition, the median values of fluorescence intensity of mast cells were converted to the numbers of the molecules of equivalent soluble fluorochrome units (MESF) using Quantum 25 microbeads (Flow Cytometry Standards, San Juan, PR) on each day that an experiment was performed, as per the specifications of the manufacturer (13).
Studies of differentiated mouse mast cells
For cell culture before flow cytometry, unfractionated peritoneal cells from adult BALB/c IL-4-/- mice (28) or the corresponding normal (IL-4+/+) BALB/c mice (Charles River Laboratory, Wilmington, MA) were cultured at 5 x 105 cells/ml in DMEM (Life Technologies) supplemented with 10% FCS, 50 ng/ml rat rSCF (Amgen), 2 mM L-glutamine, and antibiotics (penicillin/streptomycin); mast cells were incubated for up to 6 days with or without ascites containing mouse IgE anti-DNP mAb (at 5 µg/ml) (13) or mouse rIL-4 (Genzyme; at 10 ng/ml).
BALB/c bone marrow-derived cultured mast cells (BMCMCs) were generated by culturing femoral bone marrow cells of adult BALB/c mice in 20% WEHI-3 cell supernatant-conditioned medium for 4–6 wk. Mast cells were incubated for up to 4 days with or without ascites containing mouse IgE anti-DNP (at 5 µg/ml) or mouse rIL-4 (at 10 ng/ml).
For flow-cytometric analysis of unfractionated freshly isolated mouse
peritoneal mast cells, we preincubated the cells with B3B4 and 2.4G2
mAbs (PharMingen, San Diego, CA) for 15 min to block low affinity
binding of IgE or other subsequent Abs to CD23 or Fc
RII/III,
respectively (13). The cells were then incubated at 4°C with mouse
IgE anti-DNP mAb (at 10 µg/ml) for 50 min, and then,
simultaneously, for the last 25 min, with a biotinylated rat
anti-mouse c-kit Ab (at 15 µg/ml) (13). After washing,
cells were stained with FITC rat anti-mouse IgE Ab (PharMingen; at
10 µg/ml) and phycoerythrin-streptavidin (Sigma; at 14 µg/ml) for
25 min at 4°C. IgE+, c-kit+ cells
were gated and analyzed (13).
For flow-cytometric analysis of BMCMCs, cells were blocked with B3B4 and 2.4G2 as above, incubated with mouse IgE anti-DNP mAb (at 10 µg/ml) for 50 min, and then stained with FITC rat anti-mouse IgE Ab (at 10 µg/ml) (13).
Quantification of human mast cell mediator release
For measurements of mediator release, cells were sensitized with myeloma IgE at 5 µg/ml for 2 h at 37°C, washed, and stimulated with either goat anti-human IgE Ab or calcium ionophore A23187 (Sigma). For measurements of histamine, PGD2, or LTC4 release, cells were stimulated for 1 h at 37°C, and the histamine in supernatant and cell fractions was measured using a RIA kit (Immunotech, Westbrook, ME), whereas the PGD2 or LTC4 in the supernatants were measured using enzyme immunoassay kits (Cayman Chemical, Ann Arbor, MI). For cytokine release, cells were stimulated with anti-IgE for 4 h, and IL-13 in supernatants was assayed using an ELISA kit (Endogen, Woburn, MA).
Statistical analysis
Unless otherwise specified, all data are expressed as mean ± SEM, and all differences between values were compared by the two-tailed Student’s t test (unpaired).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI expression by human mast cells in vitro
As assessed by flow cytometry (see Materials and
Methods), we confirmed that baseline levels of IgE binding by
human mast cells were significantly enhanced, in a
concentration-dependent manner, upon incubation with monomeric human
IgE (18); results representative of those obtained in 12 separate
experiments, employing mast cells derived from eight different
umbilical cord blood preparations that were tested after 9–19 wk of
culture, are shown in Fig. 1
,
A and B.
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RI (13). However, to investigate this point directly, we
incubated in vitro derived human mast cells (purity = 96.3%, 9 wk
of culture) for 6 days with or without IgE (at 5 µg/ml) and then used
flow cytometry to compare the IgE-binding ability of cells, as
determined by staining with our FITC anti-human IgE Ab, with their
surface expression of FcRI, as assessed by staining with the CRA-1
Ab (27). As shown in Fig. 2,
preincubation of mast cells for 6 days with IgE markedly up-regulated
both the ability of these cells to bind IgE and their reactivity with
CRA-1. These results indicate that, most, if not all, of the
IgE-dependent up-regulation of IgE binding exhibited by these cells
reflected increased surface expression of FcRI.
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RI expression by human mast cells in vitro
We maintained aliquots of some of the same mast cells shown in
Fig. 1
for 7 days in medium containing rhSCF, rhIL-6, and 1 µM
PGE2 (i.e., our usual culture medium), or in medium
containing the same concentrations of rhSCF and rhIL-6, but without
PGE2 (20), and then for an additional 4 days in medium
containing rhSCF and rhIL-6 ± PGE2 (1 µM)
plus human rIL-4 (Genzyme; at 10 ng/ml) (20, 21), human IgE (as above,
at 5 µg/ml), or neither rhIL-4 nor IgE.
As shown in Fig. 3, incubation of mast
cells with monomeric IgE (in the presence or absence of exogenous
PGE2) resulted in a substantial enhancement of the
IgE-binding ability of cells. When compared with the most appropriate
negative control cells (i.e., cells that had not been passively
sensitized with IgE immediately before flow cytometry with FITC
anti-IgE, but that had been incubated under the same culture
conditions for the previous 4 days), the mast cells that had been
incubated with rhIL-4 expressed a slightly greater ability to bind IgE
(i.e., a somewhat greater difference in the height of the black and
grey bars in the figure) than did mast cells that had been incubated
without rhIL-4 (Fig. 3). However, any effect that IL-4 may have had on
mast cell surface expression of FcRI in this experiment was minimal
compared with that of IgE.
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RI expression of mast cells that had been incubated for 4
days before flow cytometry with rhIL-4 (10 ng/ml), monomeric IgE (5
µg/ml), human IgG (Reagent Grade, Sigma; at 5 or 100 µg/ml), or
neither Abs nor rhIL-4 (Fig. 4). As
assessed by comparison with the most appropriate negative control cells
(grey bars in Fig. 4), mast cells that had been incubated with rhIL-4
exhibited slightly enhanced IgE binding and FcRI expression (Fig. 4). Moreover, the MESF values for FcRI expression (i.e., CRA-1
binding) of cells incubated with rhIL-4 were significantly higher, by
18%, than those for cells incubated without rhIL-4,
p < 0.005 (Fig. 4). However, the effects of rhIL-4 on
the ability of the cells to bind IgE, or their surface expression of
FcRI, were much less striking than those of IgE
(p < 0.0001 for either comparison). By
contrast, incubation with IgG (at 5 or 100 µg/ml) had no detectable
effect on the FcRI expression of the cells (Fig. 4).
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RI expression, using mast cells maintained in our usual culture
medium (with PGE2, at 1 µM) and cultured for 4 days with
or without IL-4 and/or IgE (at 5 µg/ml) (Fig. 5). As assessed by flow cytometry, MESF
values for cells cultured for 4 days with IL-4 alone (at 0.1–100
ng/ml) were slightly but significantly higher than values for cells
cultured without IL-4, with the greatest enhancement of FcRI
expression by IL-4 alone occurring in cells that had been incubated
with 10 ng/ml of IL-4. By contrast, IgE alone (at 5 µg/ml) had a much
greater effect on FcRI expression than did any of the tested
concentrations of IL-4 (Fig. 5). However, when various concentrations
of IL-4 were added for the 4-day period of culture together with IgE at
5 µg/ml, IL-4 clearly exhibited a concentration-dependent synergistic
enhancement of FcRI expression compared with that induced by IgE
alone (Fig. 5).
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,
in this experiment IL-4 had little or no effect on FcRI expression
when used without IgE, but significantly enhanced FcRI expression,
over that seen in cells that had been incubated with IgE alone, when
administered together with IgE. This synergistic effect of IL-4 was
statistically significant as early as 12 h after the beginning of
the experiment, and surface FcRI expression in cells cultured with
IgE ± IL-4 continued to increase gradually with time over the
8-day period of the experiment.
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RI surface expression in eight different batches of
human umbilical cord blood-derived mast cells. Preincubation with IL-4
alone increased IgE-binding ability and/or FcRI expression
significantly, albeit minimally, in four of these experiments; in the
other four experiments, no significant effects were detected. By
contrast, IgE preincubation resulted in marked elevations of
IgE-binding ability and/or FcRI surface expression in all eight
experiments.
IgE-dependent up-regulation of human mast cell FcRI expression
can enhance IgE-dependent mast cell release of histamine and lipid
mediators, whereas IL-4 can enhance anti-IgE-dependent
mediator release without affecting levels of surface expression of
FcRI
We also assessed the potential effects of IgE-dependent
up-regulation of IgE binding (Fig. 7A) on the ability of cells to
release histamine upon stimulation with either anti-IgE or the
FcRI-independent agonist, the calcium ionophore A23187 (Fig. 7B). In confirmation of our previous findings (18), we found
that, in comparison with cells that had not been preincubated with IgE,
cells that had been preincubated for 4 days with IgE at 5 µg/ml
exhibited an FcRI-dependent histamine release response to
anti-IgE challenge that was significantly enhanced in both
sensitivity and intensity; by contrast, the histamine release response
to challenge with A23187 was not significantly affected (Fig. 7B). In a separate experiment, which was performed using
aliquots of the same mast cell preparations shown in Fig. 1B, we found that the cells that had been incubated in 5
µg of IgE/ml for 4 days gave 73% more specific histamine release
upon challenge with anti-IgE at 1 µg/ml than did cells that had
not been incubated with IgE before passive sensitization and
anti-IgE challenge.
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RI-dependent activation of human
umbilical cord blood-derived mast cells (data not shown). We then
assessed LTC4 production 1 h after FcRI-dependent
activation in a batch of mast cells (purity >90%, 10 wk of culture)
that either had or had not been preincubated with 5 µg IgE/ml for 4
days before challenge with anti-IgE. In this experiment, IgE
preincubation resulted in a 47% increase (p <
0.0001) in the MESF values of cells assessed after incubation with the
CRA-1 Ab to the FcRI -chain, and a 156% increase
(p < 0.0001) in LTC4 production by
1 h after challenge with anti-IgE at 1 µg/ml. Thus, cells
that had not been preincubated for 4 days with IgE released 0 ± 0
versus 16.7 ± 1.5 pg LTC4/104 cells after
challenge with 0 versus 1 µg/ml of anti-IgE
(p < 0.01), whereas the cells that had been
preincubated with IgE for 4 days released 0 ± 0 versus 59.5
± 0.8 pg LTC4/104 cells
(p < 0.0001).
We next tested a batch of mast cells of purity >99% (15 wk of
culture), to examine the effect of preincubation with IL-4 and/or IgE
on the FcRI expression of cells, and on their ability to release
histamine, LTC4, and PGD2 in response to
FcRI-dependent activation (Fig. 8). In
confirmation of our other experiments, a 4-day preincubation with IL-4
alone did not significantly enhance FcRI expression in these cells,
whereas FcRI expression was significantly increased by preincubation
with IgE at 5 µg/ml or, to a slightly but significantly greater
extent, by preincubation with both IgE and IL-4 (Fig. 8A).
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B,
left). Notably, a 4-day preincubation of these cells with
IL-4 at 10 ng/ml also significantly enhanced their ability to release
histamine in response to challenge with the calcium ionophore A23187 (1
µM), whereas the A23187-induced responses of cells preincubated with
IgE alone were slightly, but not significantly, less than those of
control cells that had not been preincubated with IL-4 or IgE (Fig. 8B, right). Since preincubation of these cells
with IL-4 had little or no effect on their level of surface expression
of FcRI (Fig. 8A), the effect of IL-4 preincubation on
the histamine release response of the cells to challenge with A23187
must have occurred by a mechanism independent of those related to
increased surface expression of the FcRI.
In accordance with the results for histamine release, preincubation of
mast cells with either IL-4 or IgE for 4 days enhanced the ability of
cells to release LTC4 or PGD2 in response to
anti-IgE challenge (Fig. 8C). Moreover, for either of
these lipid mediators, cells that had been preincubated for 4 days with
both IL-4 and IgE released significantly more product in response to
anti-IgE challenge than did cells that had been preincubated with
either IL-4 or IgE alone.
Finally, the data in Fig. 8 indicate that exposure of human umbilical
cord blood-derived mast cells to IL-4 can enhance the ability of these
cells to release lipid mediators (Fig. 8C) in response to
anti-IgE challenge by a mechanism that appears to be independent of
any major effect of IL-4 on the cells’ surface expression of FcRI
(Fig. 8A). Thus, pretreatment with IL-4 alone had no
significant effect on the cells’ surface expression of FcRI (Fig. 8A), but did significantly enhance the ability of cells to
release LTC4 (Fig. 8C, left) or
PGD2 (Fig. 8C, right), as well as
histamine (Fig. 8B), upon anti-IgE-dependent activation.
Different batches of umbilical cord blood-derived human mast cells can vary markedly in their mediator release responses to anti-IgE challenge after preincubation with or without IgE and/or IL-4
We then performed an experiment similar to that depicted in Fig. 8, but employing a different batch of cord blood-derived mast cells and
including a dose-response examination of the effects of various
concentrations of anti-IgE on LTC4 and PGD2
release, as well as on histamine release (Fig. 9). In this experiment, preincubation
with IL-4 alone slightly, but significantly, enhanced surface
expression of FcRI, whereas preincubation with IgE alone had a much
greater effect (Fig. 9A). However, the synergistic effect of
IL-4 and IgE preincubation of FcRI surface expression was greater in
these mast cells than in those that were employed for the studies shown
in Fig. 8 (cf Figs. 8A and 9A).
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appeared to be substantially less responsive to anti-IgE-induced
release of histamine or lipid mediators than were the cells used for
the experiments shown in Fig. 8. For example, minimal specific release
of histamine, LTC4, and PGD2 (white circles in
Fig. 9, B and C, respectively) was induced by
anti-IgE challenge of cells that were preincubated with neither IgE
nor IL-4 before short-term incubation with IgE before anti-IgE
challenge. However, the anti-IgE-induced release of all three
mediators was significantly increased, versus values for cells
preincubated without IgE or IL-4, in cells that had been preincubated
with either IgE or IL-4 alone, and the enhancing effects of the
preincubation with IL-4 alone were the same as (for PGD2
release, Fig. 9C) or even greater than (for histamine, Fig. 9B, or LTC4, Fig. 9C) those observed
with cells that were preincubated with IgE alone. The enhancing
effects, on anti-IgE-induced mediator secretion, of preincubation
with both IL-4 and IgE were greater in the cells shown in Fig. 9 than
in those shown in Fig. 8, particularly in comparison with the effects
observed in cells that were incubated with IgE alone.
Analysis of results obtained with several different batches of
umbilical cord blood-derived human mast cells provides further support
for the conclusion that such cells can exhibit considerable
batch-to-batch variation in the magnitude of their mediator release
responses to anti-IgE challenge, as well as in their patterns of
responsiveness to anti-IgE challenge after preincubation with or
without IgE and/or IL-4. Thus, we have now analyzed the effects of
preincubation with IgE alone on anti-IgE-induced histamine release
in two experiments, and have compared the effects of preincubation with
IgE alone, IL-4 alone, or both IgE and IL-4 on anti-IgE-induced
histamine release in five experiments, for a total of seven
experiments, each employing a different batch of cord blood-derived
mast cells. In two of these experiments (those depicted in Figs. 7 and 8), anti-IgE challenge induced >10% specific histamine release in
cells that had been preincubated with neither IgE nor IL-4, whereas in
five experiments (including that depicted in Fig. 9), anti-IgE
challenge induced <10% specific histamine release from such control
cells.
Although we have performed relatively few studies of
anti-IgE-induced release of lipid mediators, the three different
mast cell populations analyzed exhibited large differences in their
responses. Thus, in the cells used for the experiments shown in Fig. 8, preincubation with IgE alone had a substantial enhancing effect on the
ability of cells to release LTC4 in response to
anti-IgE at 1 µg/ml. By contrast, in the experiments shown in
Fig. 9, cells preincubated with or without IgE released very small, and
quite similar, amounts of LTC4 in response to anti-IgE
(at either 1 or 10 µg/ml).
Moreover, the variation in the ability of different batches of mast
cells to produce mediators in response to anti-IgE challenge
appeared to be greater in the case of some products than others. For
example, the cells examined in Figs. 8 and 9 were much more similar in
their ability to release PGD2 in response to challenge with
anti-IgE at 1 µg/ml than they were in their ability to produce
LTC4 under the same conditions.
Such differences in responses may reflect differences in baseline
levels of FcRI surface expression (in control cells that were not
preincubated with IgE or IL-4) and/or multiple other factors.
Notably, there was less batch-to-batch variation in the responses of
these mast cell populations to the effects of preincubation with IgE,
IL-4, and/or IL-4 on anti-IgE-induced histamine release. In each of
the five experiments of this type, the effects of preincubation with
IgE or IL-4 alone were significantly less than those of preincubation
with IgE plus IL-4. Moreover, we found that in high responder mast
cells, defined as those that gave >10% specific histamine release
upon anti-IgE challenge after preincubation for 4 days without IgE
or IL-4, the effect of adding IL-4 to IgE for a 4-day preincubation was
small, whether judged by levels of FcRI expression or magnitude of
anti-IgE-induced histamine release. By contrast, adding IL-4 to IgE
for a 4-day preincubation generally resulted in substantially greater
enhancement of FcRI surface expression or anti-IgE-induced
histamine release in the low responder cells (defined as those that
gave <10% specific release of histamine upon anti-IgE challenge
after preincubation for 4 days without IgE or IL-4).
Additional studies will be required to ascertain the mechanism(s) underlying the effects of IL-4 on mediator secretion by umbilical cord blood-derived human mast cells. Nevertheless, in the case of both histamine release and PGD2 or LTC4 production, the levels of anti-IgE-induced mediated release observed in cells preincubated with both IL-4 and IgE significantly exceeded those seen in cells preincubated with IgE alone.
Effects of IgE and/or IL-4 on FcRI surface expression on
differentiated mouse mast cells
A preliminary report indicated that, under certain circumstances,
IL-4 can suppress FcRI surface expression on mouse mast cells in
vitro; however, the specific details of the conditions for testing IL-4
in these experiments were not described (29). To examine the effects of
IL-4 preincubation on FcRI surface expression on differentiated
mouse mast cells, we first tested BALB/c BMCMCs, representative of
relatively immature in vitro derived mouse mast cells, which had been
preincubated for 4 days before flow cytometry with mouse IgE mAb (5
µg/ml), mouse rIL-4 (10 ng/ml), both IgE (5 µg/ml) and IL-4 (10
ng/ml), or neither IgE nor IL-4. As shown in Fig. 10, preincubation with IgE alone
resulted in a marked increase in FcRI expression in these cells (in
confirmation of previous reports) (13, 14), whereas IL-4 had no
significant effect, whether when used alone or in conjunction with IgE.
|
RI surface expression at baseline
(MESF = 0.55 ± 0.04) than did PMCs from IL-4+/+
mice (1.87 ± 0.25, p < 0.001 versus values for
IL-4-/- PMCs) (Fig. 11).
This finding probably reflected the lower levels of circulating IgE in
the IL-4-/- mice (13). Indeed, in mice that had been
injected i.v. with mouse IgE mAb (100 µg/mouse, i.v., for each of 4
days before recovery of PMCs for flow cytometry) (13), MESF values for
FcRI surface expression by PMCs were statistically indistinguishable
in IL-4-/- versus IL-4+/+ PMCs (1.84 ±
0.09, n = 4 versus 1.93 ± 0.07, n
= 5; p >0.05).
|
RI surface expression in both IL-4-/- and
IL-4+/+ PMCs, an effect that, in the case of the
IL-4-/- PMCs, must have been entirely independent of
IL-4. By contrast, incubation with exogenous IL-4 alone had little or
no effect on FcRI surface expression. However, for PMCs derived from
either IL-4+/+ or IL-4-/- mice, MESF values
for FcRI surface expression were significantly higher in cells that
had been incubated with IL-4 and IgE than in those that had been
incubated with IgE alone. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI. In confirmation of prior studies (20, 21, 22), we
also found that, in some experiments, incubation of human mast cells
with IL-4 resulted in a modest enhancement of their surface expression
of FcRI. However, in our in vitro derived human mast cell
preparations, unlike the human fetal liver-derived mast cells in
earlier stages of development that were studied by Xia et al. (22),
monomeric IgE exhibited a much more striking ability to up-regulate
mast cell surface expression of FcRI than did IL-4 alone. We
obtained very similar findings with two different populations of
differentiated mouse mast cells, in vitro derived BMCMCs and in vivo
derived PMCs. On the other hand, IL-4 and IgE acted synergistically in
umbilical cord blood-derived human mast cell populations (and in mouse
peritoneal mast cells) to enhance FcRI surface expression, findings
that are consistent with the results reported by Xia et al. (22) for
fetal liver-derived human mast cells.
The differences between some of our results and those of Xia et al.
(22) may largely reflect differences in the mast cells that were
analyzed in the two studies. We performed our experiments comparing the
effects of IgE and IL-4 on FcRI expression using differentiated mast
cells that were obtained by maintaining unbilical cord blood
mononuclear cells for at least 9 wk in medium supplemented with SCF,
IL-6, and PGE2 (24). By contrast, the recent report by Xia
et al. (22) (which was published after many of the experiments reported
herein had been completed) utilized fetal liver-derived human mast
cells that were generated in SCF-containing medium, but were
investigated only during the first 4 wk of their development in vitro.
Moreover, in the fetal liver-derived mast cell system, the effects of
IL-4 on FcRI expression were greatest when the cytokine was present
during the earliest stages (i.e., the first 2 wk) of mast cell
development in vitro (22).
Accordingly, one possible explanation for the differences between our
results and those of Xia et al. (22) is that, in mast cells
that have undergone more extensive differentiation/maturation in vitro,
FcRI expression becomes less responsive to IL-4 and more responsive
to IgE. If this hypothesis is correct, it raises the possibility that
the same may be true in vivo, i.e., that IL-4 is important in promoting
FcRI expression during early stages of mast cell development,
through either direct or indirect effects on mast cells, but that IgE
becomes more important than IL-4 as a regulator of FcRI expression
at later stages of mast cell differentiation/maturation. Alternatively,
some of the differences obtained in the two studies may reflect
intrinsic differences between umbilical cord blood-derived as opposed
to fetal liver-derived human mast cells.
The effects of IL-4 on FcRI expression may represent one component
of a wide spectrum of actions of this cytokine (when tested in
concentrations similar or identical to those used in this study) on
mast cell development and function. For example, in human umbilical
cord blood-derived mast cells generated in medium containing rhSCF and
rhIL-6, exposure of the cells to IL-4 at 10 ng/ml induced morphologic
changes consistent with enhanced mast cell maturation, and also
increased the cells’ expression of chymase, changes that were apparent
as early as 5 days after the first exposure to exogenous IL-4 (31). In
mouse mast cells, exposure to IL-4 not only enhanced the ability of
cells to release serotonin in response to challenge with IgE and
specific Ag, but also increased the serotonin release of the cells in
response to challenge with compound A23187 (32). Exposure to IL-4 also
can enhance the ability of in vitro derived mouse mast cells to release
histamine and LTC4 in response to endothelin-1 (33).
It seems very unlikely that the effects of IL-4 on mast cell secretion
of mediators in response to compound A23187 (this study and 32) or
endothelin-1 (33) would be due to any effects of the cytokine on
surface expression of FcRI. Indeed, in the present study, it is
possible that the enhanced anti-IgE-induced release of histamine
and lipid mediators observed in cells that had been preincubated with
IL-4 and IgE, as opposed to IgE alone (Figs. 8 and 9), reflected a
combination of the effects of IL-4 exposure on both FcRI surface
expression and on other, FcRI-independent, aspects of mast cell
maturation and secretory function.
While the precise mechanisms underlying the spectrum of effects of IL-4
on human mast cell mediator secretion remain to be fully elucidated,
increasing evidence indicates that exposure to monomeric IgE can
enhance IgE-dependent mast cell mediator release largely, if not
entirely, by up-regulating levels of surface expression of the FcRI.
Thus, IgE-dependent up-regulation of human mast cell FcRI expression
can significantly enhance the sensitivity and/or intensity of the
response of cells to anti-IgE challenge, as reflected by the
secretion of increased quantities of histamine (this study and 18), LTC4 and PGD2 (this study), MIP-1 (18),
or tryptase (22). Moreover, our preliminary studies indicate that
IgE-dependent up-regulation of FcRI surface expression can also
enhance the ability of umbilical cord blood-derived human mast cells to
secrete IL-13 (our unpublished data).
Our findings suggest that any mechanism that results in the substantial
elevation of IgE levels is most likely also to result in significantly
enhanced IgE-dependent human mast cell function. IgE can also regulate
surface FcRI expression by human (34) or mouse (35) basophils,
raising the possibility that circulating or local concentrations of IgE
can enhance FcRI expression, and FcRI-dependent function, in
other cell types as well. For example, it will be of interest to
determine whether this mechanism can account, at least in part, for
observations indicating that subjects with atopy may express increased
levels of FcRI on CD1a+ Langerhans cells (36) or
circulating monocytes (37), or for the apparent differences in the
levels of FcRI expression in various populations of eosinophils
(38). Furthermore, no matter what minimum number of cross-linked
FcRI may be required to elicit functional responses in
FcRI+ effector cells (39, 40), cells that express
increased capacity for IgE binding on their surface can be sensitized
adequately with larger numbers of different IgE species of distinct Ag
specificities.
Our findings strongly support the hypothesis that approaches that
significantly diminish circulating levels of IgE have the potential to
reduce the IgE-binding capacity of tissue mast cells, as well as
circulating basophils (34, 41), and that this, in turn, can diminish
IgE- and FcRI-dependent mast cell effector function. Indeed, this
represents one of the more likely explanations for the observation that
subjects in whom serum levels of IgE had been markedly reduced as a
result of treatment with an anti-human IgE Ab also exhibited
diminished wheal and flare reactions in response to intradermal
allergen challenge (34).
Finally, we found that individual batches of umbilical cord
blood-derived human mast cells exhibited wide variation in their
ability to release histamine in response to anti-IgE challenge,
particularly if the cells had not been preincubated with IgE for 4 days
before further sensitization with IgE just before challenge with
anti-IgE. Based on the results shown in Figs. 8 and 9, it appears
that mast cells may also exhibit considerable variation in their
ability to release lipid mediators in response to anti-IgE
challenge. By contrast, less variation was observed in the ability of
different populations of mast cells to undergo up-regulation of FcRI
surface expression in response to preincubation for 4 days with IgE.
Because each cord blood preparation was derived from a genetically distinct, albeit unidentified, donor, the variation in the responses of different populations of umbilical cord blood-derived mast cells to anti-IgE challenge may have reflected genetic differences in the donors. It is even possible that some of these genetic differences may also influence the susceptibility of these individuals to the development of allergic diseases. However, there are many other factors that may have contributed to these results, including differences in the responses of individual cord blood cell preparations to our conditions of culture.
While extensive further studies may be needed to understand the basis for these observations, the potential variability of the responses of different cord blood-derived human mast cell populations to challenge with anti-IgE should be kept in mind whenever the results obtained with such mast cell populations are analyzed.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Division of Allergology and Rheumatology, Department of Medicine, University of Tokyo, School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
3 M.Y. and K.S. are co-first authors.
4 Current address: First Department of Internal Medicine, Nagasaki University, School of Medicine, 1-7-1 Sakamoto, Nagasaki 852, Japan.
5 Address correspondence and reprint requests to Dr. Stephen J. Galli, Department of Pathology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5324. E-mail address:
6 Abbreviations used in this paper: MIP-1, macrophage-inflammatory protein-1; BMCMCs, mouse bone marrow-derived cultured mast cells; h, human; LTC, leukotriene C; MESF, molecules of equivalent soluble fluorochrome units; PMCs, mouse peritoneal mast cells; rhSCF, recombinant human stem cell factor 164; SCF, stem cell factor.
Received for publication August 14, 1998. Accepted for publication February 8, 1999.
|
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10%). *, p 
, No pretreatment; 
, IL-4 at 10 ng/ml; •, IgE at
5 µg/ml; 
, both IgE at 5 µg/ml and IL-4 at 10 ng/ml. *,
p < 0.05; **, p < 0.001;
***, p < 0.0001, versus corresponding values
for mast cells that had been cultured for 4 days without IgE or IL-4.
C, Left, LTC4 secretion;
right, PGD2 secretion. White bars, no
pretreatment; stippled bars, IL-4 at 10 ng/ml; striped bars, IgE at 5
µg/ml; black bars, both IgE at 5 µg/ml and IL-4 at 10 ng/ml.
*, p < 0.05; **, p <
0.001; ***, p < 0.0001, versus corresponding
values for mast cells of the same pretreatment group, but that had not
been stimulated with anti-IgE.
