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The Johns Hopkins University School of Medicine, Division of Clinical Immunology, The Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The concept that basophils play an active role in allergic disease has
intensified in recent years with evidence that these cells secrete
immunomodulatory cytokines (5). In particular, mRNA for IL-4 is
up-regulated within 30 min after IgE-dependent activation and is
followed by the secretion of high levels of protein for this cytokine
(6). Basophils appear to be the sole source of IL-4 protein secreted in
mixed leukocyte cultures, even when challenged with specific Ag (6, 7).
Furthermore, they produce this cytokine in a time frame (1–4 h) that
is consistent with the kinetics of the late phase response to allergen
exposure. In addition to IL-4, several laboratories have now confirmed
that human basophils also produce IL-13 following cellular activation
(8, 9, 10), and one study has reported the production of macrophage
inflammatory protein-1
by these cells (11).
It is of considerable interest that human basophils generate and secrete both IL-4 and IL-13 protein, since these cytokines are reported to share many biologic properties that are central in allergic inflammation (12). These include 1) Ig isotype switching in B lymphocytes from IgM to IgE, 2) the up-regulation of CD23 and HLA class II molecules on B lymphocytes, and 3) the promotion of increased expression of vascular cell adhesion molecule-1 on endothelial cells, an adhesion protein regulating the selective transendothelial migration of eosinophils, lymphocytes, and basophils (13, 14, 15, 16). There are, however, differences in the functional activities of these two cytokines. Notably, IL-13 does not appear to modulate T cell function, while IL-4 induces the differentiation of the Th2 phenotype (17, 18) and regulates CD8+ T cell functions (19).
As noted above, IgE-dependent activation triggers the release of both IL-4 and IL-13 from basophils. The generation of these cytokines does not appear to occur in tandem, as recent reports show that IL-13 secretion starts at a time (i.e., 4 h) when IL-4 production is near maximal and peaks some 24 h following stimulation (9, 10). The aim of this study was to examine the relationship between IL-4 and IL-13 secretion from basophils by comparing a variety of parameters. Our results show that there is no correlation between the magnitude of IL-13 and IL-4 secretion among donors cells. Further, the differential effects of various secretogogues on the induction of these cytokines and evidence that their synthesis is pharmacologically independent from one another suggest that distinct intracellular pathways control IL-4 and IL-13 secretion from basophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following special reagents were purchased: biotinylated goat anti-human IL-4 (Endogen, Irvine, CA); crystallized BSA, FBS, PIPES, and ionomycin (Sigma Chemical Co., St. Louis); crystallized human serum albumin, PMA, and bisindolylmaleimide II (BIS II;3 Calbiochem-Behring Corp., La Jolla, CA); EDTA, D-glucose, Tween-20, and Tween-80 (Fisher, Raleigh, NC); FK506 (Fujisawa USA, Deerfield, IL); gentamicin, Iscove’s modified Dulbecco’s medium (IMDM) containing L-glutamine and 25 mM HEPES, RPMI 1640 with 25 mM HEPES and L-glutamine, and nonessential amino acids (100x stock; Life Technologies, Inc., Grand Island, NY); 60% perchloric acid (Fisher Scientific, Fairlawn, NJ); and Percoll (Pharmacia Biotec, Inc., Piscataway, NJ).
The polyclonal anti-human IgE Ab used in these studies was made in a goat (20). Recombinant human IL-4, recombinant human IL-3, and monoclonal anti-human IL-4 Ab were gifts of Steven Gillis from Immunex Corp. (Seattle, WA).
Buffers and media
PIPES (10x) contained 250 mM PIPES, 1.10 M NaCl, and 50 mM KCl, pH 7.4, and was stored at 4°C as a stock solution. PAG contained 1/10th 10x PIPES in addition to 0.003% human serum albumin and 0.1% D-glucose. PAG-EDTA additionally contained 4 mM EDTA. Isotonic Percoll (referred to in this manuscript as 100% Percoll) was prepared by mixing 1 part 10x PIPES and 9 parts Percoll. Percoll solutions of 50% (density = 1.066), 55% (density = 1.072), 60% (density = 1.079), and 62% (density = 1.082) were made with the appropriate combinations of 1x PIPES and 100% Percoll. Conditioned IMDM (C-IMDM) consisted of IMDM supplemented with 5% heat-inactivated (56°C, 30 min), FBS, 1x nonessential amino acids, and 5 µg/ml of gentamicin.
Preparation of basophil-enriched mixed leukocyte suspensions
Venous blood from consenting adults was anticoagulated in 10 mM EDTA, then centrifuged at 300 x g. The resulting buffy coat at the leukocyte/RBC interface was aspirated and combined with PAG-EDTA (1/1, v/v). Basophil-enriched leukocyte suspensions (5–50% basophils) were prepared as previously described (21). Briefly, the leukocyte mixture was layered onto a double Percoll gradient consisting of 12 ml of 55% Percoll layered onto 12 ml of 62% Percoll in 50-ml polypropylene centrifuge tubes (Corning, Corning, NY). The Percoll gradients were centrifuged at 700 x g for 20 min at room temperature. Basophils were concentrated in the lower half of the 55% Percoll fraction, at the 62% interface and the upper half of the 62% fraction. The basophil-rich fraction was harvested and washed twice in PAG-EDTA, once at room temperature and then at 4°C. A third wash was performed using PAG at 4°C. Basophil purity and number were determined using a Spiers-Levy chamber after staining with Alcian blue (22).
Preparation and purification of basophils from leukopheresis packs
Peripheral blood basophils from leukopheresis sediments were isolated and purified using a combination of countercurrent elutriation and Percoll density centrifugation as previously described (6). Briefly, leukopheresis sediments were layered onto a 65% (density = 1.083) Percoll gradient and centrifuged at 400 x g for 20 min. The mononuclear cell interface was collected and loaded into an elutriation chamber (model J2-21, Beckman, Palo Alto, CA) at a constant flow rate of 14 to 15 ml/min with a rotor speed of 3600 rpm. Five fractions of leukocytes were collected by decreasing the speed of the rotor. After collecting the initial fraction at 3600 rpm, the rotor speed was reduced sequentially to 3000, 2550, and 2280 rpm. Basophils were most often found in elutriation fractions 3 and 4. Cells in these fractions were recovered by centrifugation, then layered on a double Percoll gradient consisting of densities of 1.066 and 1.079. After this step, leukocyte suspensions of purity ranging from 10 to 30% were obtained. After an overnight culture in RPMI 1640 supplemented with 2% FBS and 10 µg/ml gentamicin, basophils were purified up to 93% on a second double Percoll gradient (densities = 1.069/1.079).
Culture conditions
For all experiments, basophils were cultured for cytokine
secretion and histamine release in C-IMDM using protocols previously
established in our laboratory (6, 21, 23, 24, 25). Experiments were
conducted in 96-well flat-bottom microtiter plates (Costar, Cambridge,
MA), with the number of basophils varying from 50,000 to 420,000/well.
Each condition tested was performed in duplicate. For secretion
studies, cells were added to culture wells in 125 µl of medium and
preincubated for 15 min (37°C, 5% CO2 incubator).
The stimulus (at twice the final concentration) or medium was added
(125 µl), and after 4 or 20 h, the supernatants were harvested
by centrifugation of microtiter plates (150 x g, 3
min). For histamine analysis, the top 25 to 50 µl of supernatant from
the 4-h cultures were carefully removed and transferred to
corresponding tubes containing 1 ml of PAG with 1.6%
HClO4. After an overnight precipitation of protein at
4°C, the samples were centrifuged, and the histamine content in the
supernatants was assayed by automated fluorometry (26). The percent
histamine secretion was calculated from total histamine content
determined by lysing the proportion of basophils equivalent to that
occurring in the amount of supernatant used for histamine measurements.
Basal release of histamine (i.e., release in medium alone), even after
4 h of culture, was <15%. The remaining culture supernatants
(
190 µl each) were frozen at -80°C for IL-4 and IL-13 protein
measurements determined by ELISA. For studies testing the effects of
FK506 and BIS II on basophil secretion, the inhibitor and stimulus were
added to cultures simultaneously.
Cytokine gene expression studies, RNA isolation, and RT-PCR
Mixed leukocyte suspensions containing 12 to 25% basophils were preincubated (37°C, 5% CO2) in autoclaved (RNase-free) 1.5-ml polypropylene microcentrifuge tubes at a total cell concentration of 4 to 8 x 106/ml in 0.25 to 0.5 ml of C-IMDM. Cells were then challenged with an equal volume of medium alone, FK506, anti-IgE/IL-3, or the combination of anti-IgE/IL-3 and FK506. After 2 and 4 h, the cell cultures were centrifuged (12,000 x g) for 10 s, and the supernatants were removed for IL-4 measurements as described above. Total RNA was extracted from the cell pellets using the RNAzol protocol (Tel-Test, Inc., Friendswood, TX). Following isopropanol precipitation, the RNA was washed with 70% ethanol and dried in a speed vacuum centrifuge. Subsequently, the RNA was resuspended in 25 µl of diethylpyrocarbonate-treated water and stored at -80°C. It is important to note that fourfold dilutions of sample RNA were made in sterile diethylpyrocarbonate-treated H2O before RT-PCR. This was performed to better determine (qualitatively) the relative amounts of mRNA among the samples. RT-PCR was performed as previously detailed (6) using the GeneAmp RNA PCR kit (Perkin-Elmer/Cetus, Norwalk, CT) according to the manufacturer’s instructions. Briefly, first strand cDNA was synthesized from an aliquot of RNA in the presence of murine leukemia virus reverse transcriptase (2.5 U/µl); 1 mM each of dATP, dCTP, dGTP, and dTTP; RNase inhibitor (1 U/µl); 5 mM MgCl2; and PCR buffer (50 mM KCl and 10 mM Tris-HCl), using oligo(dT)16 as a primer. The mixture was incubated at 42°C for 15 min in a Perkin-Elmer/Cetus thermocycler followed by 5 min at 95°C. PCR amplification was performed on aliquots of the cDNA in the presence of MgCl2, dNTPs (0.4 mM each), AmpliTaq polymerase (1 U/20 µl of reaction volume), and paired specific primers (0.2 µM of each) for hypoxanthine phosphoribosyl transferase (HPRT), IL-4, and IL-13. PCR conditions were as follows: initial denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s. HPRT was cycled 30 times, and IL-4 and IL-13 were cycled 25 times before the final extension at 72°C for 10 min. HPRT and cytokine primers were synthesized at the Johns Hopkins DNA Synthesis Laboratory. For IL-4, these represented primers in exons 1 and 4 of the genomic sequence: IL-4: 5' primer, 5'-ATG GGT CTC ACC TCC CAA CTG CT; 3' primer, 5'-GTT TTC CAA CGT ACT CTG GTT GGC; IL-13: 5' primer, 5'-GGA AGC TTC TCC TCA ATC CTC TCC TGT T; 3' primer, 5'-GCG GAT CCG TTG AAC CGT CCC TCG CGA AA; HPRT: 5' primer, CGA GAT GTG ATG AAG GAG ATG G; 3' primer, GGA TTA TAC TGC CTG ACC AAG G. Two distinct bands for IL-4 were observed on agarose gels using the above protocol, and this phenomenon has been previously noted (6, 24). A dominant band was observed with a size of approximately 460 bp. The source of the smaller, fainter band is unknown, but is thought to be an alternatively spliced form of IL-4. The neat product and dilutions were electrophoresed on agarose gel (1%) and visualized by ethidium bromide staining.
Cytokine determination using ELISA
Culture supernatants were stored at -80°C until analysis. IL-4 measurements were performed using an in-house ELISA, as previously described (25). The sensitivity of the assay was consistently 4 pg/ml with a range up to 200 pg/ml. IL-13 measurements were made using IL-13 ELISA kits (Biosource International, Camarillo, CA). The commercial ELISA was performed according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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a, the ionomycin-stimulated
IL-13 was found to correlate significantly with basophil purity
(p = 0.007), thus indicating that the
contribution of the other contaminating leukocytes was negligible.
Using IL-3 (100 ng/ml) as the stimulus, the secretion of IL-13 in the
same leukocyte preparations also significantly correlated with basophil
purity (Fig. 1b; p = 0.03), although the
levels detected were greatly reduced compared with those obtained using
ionomycin. These findings are similar to our previous observations that
IL-4 production is strictly a function of basophil purity (6). Thus, in
subsequent experiments we focused on using basophil-enriched
suspensions rapidly prepared by Percoll centrifugation, since cells
isolated in this manner are more responsive to physiologic
stimulation (21).
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a clearly show that IL-13
production from basophils is unrelated to cellular activation as
assessed by histamine secretion (p = 0.042).
This disparity is underscored by the finding that IL-4 secretion from
the same donors’ cells does highly correlate with histamine release
(p = 0.007; Fig. 2b).
Finally, a comparison of the levels of IL-13 and IL-4 protein generated
in these cultures showed that there was no correlation between the two
secretory events (Fig. 2c; p = 0.49). Cells
secreting high levels of one cytokine do not necessarily produce large
quantities of the other.
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a,
IL-3 alone caused IL-13 secretion, while not stimulating significant
IL-4 production in the same cell preparations even after 20-h
incubation, suggesting that IL-4 and IL-13 secretion are differentially
regulated. It is important to note that IL-4 protein also was not
detected in culture supernatants examined at 4 h or at a time
optimal for the secretion of this cytokine using IgE-dependent stimuli
(data not shown). We found that the addition of anti-IgE did not
significantly enhance IL-13 secretion induced by IL-3, while, as
expected, the combination of IL-3 and anti-IgE did induce the
secretion of IL-4. However, when used alone, anti-IgE Ab did induce
IL-13 protein levels (134 ± 73; n = 10), but only
in approximately 50% of the donor cells tested (data not shown).
Figure 3b shows the percentage of histamine released in the
corresponding cell cultures after 4-h incubation. IL-3 had little
effect on histamine release when used alone, yet the combination of
IL-3 and anti-IgE induced significant histamine release.
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, addition of anti-IgE increased the
rate of IL-13 induced by the IL-3 (by 4 h between the 4–12 h
points), but did not affect the quantity eventually released after 20-h
incubation. Thus, IL-13 protein was first detected after 4-h incubation
with the combination of IL-3/anti-IgE and after 8 h with IL-3
alone.
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a, PMA alone (at 1
ng/ml), in fact, was a potent secretogogue for IL-13, generating levels
about 60% of those induced with the ionomycin control. In these same
supernatants, marginal levels of IL-4 protein were detected. We did
observe that PMA, when used at <1 ng/ml or at higher amounts (10 and
100 ng/ml), induced less IL-13 protein while also failing to generate
IL-4 (Fig. 5a, inset). Furthermore, it is unlikely that the
contaminating leukocytes (7–17%) in these highly enriched basophil
cultures contributed to the PMA-induced IL-13, since subsequent
experiments showed that levels of this cytokine also correlate well
with the presence of basophils (Fig. 5b).
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). In contrast, IL-13
secretion was unaffected by this drug. Note that measurements of IL-4
and IL-13 protein were made after 4 and 20 h, respectively, or at
times optimal for the secretion of each cytokine. We also tested IL-4
secretion after 20 h and observed similar inhibition, suggesting
that FK506 does not simply alter the kinetics of IL-4 secretion (data
not shown). From these data, it appears that an FK506-insensitive
pathway exists for IL-13 secretion from basophils. In fact, the data
listed in Table I show that IL-3 induced
signals for IL-13 generation that were unaffected by FK506, with a
protein level that averaged 373 ± 93 pg/106 basophils
in the absence of FK506 and 389 ± 102 pg/106 in the
presence of 10 ng/ml of drug. In these same experiments, the IL-13
induced by anti-IgE alone (226 ± 45 pg/106
basophils) was inhibited some 70% by FK506. Likewise, the IL-4
generated (325 ± 73 pg/106 basophils) was also
inhibited by some 93%.
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, cultures treated with
IL-3/anti-IgE clearly showed an accumulation of IL-4 and IL-13
message after 4 h of incubation. However, only IL-4 message
appeared to be affected by the addition of FK506, inhibiting its
accumulation about 75% when comparing the intensities of the bands
seen with the various dilutions. Thus, the intensity of the IL-4 cDNA
product generated from undiluted RNA isolated from cells treated with
FK506 (Fig. 7, lane 13) is approximately the same as
that generated from RNA isolated from cells receiving stimulus alone
and diluted 1/4 (lane 10). IL-13 mRNA from
these same cultures appear unchanged. Similar results were seen at
2 h, but the overall levels of IL-13 mRNA were too low for
comparison at this time point (data not shown).
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indicate that
FK506 did not significantly inhibit PMA-induced IL-13 secretion when
used at concentrations between 0.08 and 10.0 ng/ml. In contrast, FK506
dramatically attenuated the IL-13 induced by ionomycin, in a
dose-dependent manner (IC50 = 200 pg/ml). These data
suggest that multiple pathways have a role in the secretion of IL-13,
at least one that is calcium dependent and one that is controlled
through PKC activation.
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, the compound, BIS II, a selective PKC
inhibitor (28), dose-dependently inhibited the IL-13 induced by PMA
alone with an IC50 of 1 µM. In contrast, at this same
concentration of drug there was no inhibition of the IL-13 induced by
either IL-3 or anti-IgE Ab, and in fact, it appeared to produce
some enhancement, although this was not significantly different from
the control value.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI receptor than is IL-4 secretion. We extended the study to
compare the kinetics of IL-13 secretion induced by IL-3 alone. Although
IL-13 secretion was considerably slower (4 h) with this stimulus, the
levels of cytokine detected after 20 h were essentially the same
as those induced with the combination of IL-3 and anti-IgE. These
findings imply that IL-3 acts as a complete stimulus for basophil IL-13
secretion. Further, we found that IL-3 did not cause histamine
secretion in the same cultures that produced IL-13. Thus, we have shown
that IL-13 production from basophils can be dissociated from histamine
secretion, and therefore, it is possible that it may be produced in
vivo without the concomitant secretion of either IL-4 or histamine.
These findings would also suggest that although IL-4 and IL-13 share a
number of biologic activities, the overall functions of these cytokines
in vivo are likely to be quite different. Their overlapping actions, in
fact, are thought to be a consequence of the existence of shared
receptor components (30). In terms of allergic disease, the most
important shared action of IL-4 and IL-13 is their ability to promote
IgE synthesis by B cells (31, 32). The levels of IgE produced in vitro
in response to IL-13 are generally lower than those obtained with IL-4
(31), and it has been hypothesized that IL-13 has an important role in
the regulation of enhanced (or ongoing) IgE synthesis in allergic
individuals (31). Since basophils express CD40L (33), and this protein
is an important costimulus required by B cells for IgE synthesis,
basophils may help maintain the production of this Ig in the periphery
of allergic subjects. Our studies, like those reported previously (9, 10), showing that basophil secretion of IL-13 is much slower than that
of IL-4, could indeed support the hypothesis that IL-13 enhances and
sustains that production of IgE in the absence of IL-4. Finally, it may
be pertinent that we did not find any correlation between either IL-13
and IL-4 levels or between IL-13 and histamine levels in our basophil
cultures, because this is compatible with the concept that IL-13 has a
homeostatic role in B cell IgE synthesis, possibly having a role in
maintaining nonspecific IgE levels. Our findings also indicate that basophil-derived IL-13 could, in certain instances, be initiated at sites not associated with immediate hypersensitivity reactions. Pertinent to this, it has been shown since the 1970s that basophils infiltrate specific delayed hypersensitivity lesions, commonly referred to as cutaneous basophil hypersensitivity reactions (34). Most clinically relevant are the cutaneous basophil hypersensitivity reactions in the human skin following exposure to rhus toxoid (35) and in chronic granulomatous conditions, such as that associated with Crohn’s disease (36).
Most strikingly, our results suggest that IL-4 and IL-13 secretion are
differentially regulated in response to PKC activation induced by
phorbol esters. In fact, we found PMA to be a potent activator of IL-13
and, quite remarkably, a very specific stimulus, since its production
correlated well with the presence of basophils in mixed leukocyte
cultures. The optimal concentration of PMA required for IL-13 secretion
was approximately 1 ng/ml, while this response declined (by nearly
50%) at higher concentrations. In previous work we showed that PMA
down-regulates IL-4 secretion in basophil cultures induced by ionomycin
(27). In the same study PMA synergized with ionomycin to promote the
secretion of this cytokine in lymphocyte cultures. In light of the
evidence presented here, we hypothesize that PKC activation is pivotal
in the regulatory mechanisms controlling IL-13 secretion from basophils
and, conversely, has a negative role in IL-4 production. The molecular
mechanism for such control may be partially unraveled by recent studies
using a Jurkat T cell clone transfected with a construct of the human
IL-4 promoter (37, 38). In this model, PMA was shown to activate the
transcription factor NF-
B, which decreased IL-4 transcription by
competing with a second factor, NF-AT, for binding sites within the
promoter. It is possible that the competitive nature of these
transcription factors may have an important regulatory role in the
generation of IL-4 and IL-13 in basophils. Preliminary evidence for
NF-AT involvement in FcRI activation has come from recent studies
using rodent cells (39). Further, we have found that a protein,
immunologically identical with NF-AT, is resident in the cytosol of
basophils and is also translocated to the nucleus within 5 min of
IgE-dependent activation (J. T. Schroeder, unpublished
observations). Therefore, NF-AT may control the latter steps in the
signal transduction pathway linking FcRI engagement with the
expression of IL-4.
In comparing the pharmacologic control of IL-4 and IL-13 secretion, we found additional evidence to support the belief that signal transduction pathways exist in human basophils that differentially regulate IL-13 and IL-4 generation in these cells. First, we found that the immunosuppressant, FK506, was a potent inhibitor of IL-4 secretion from basophils stimulated with the combination of IL-3 and anti-IgE. Likewise, the accumulation of mRNA for this cytokine was also dramatically inhibited with this drug, as assessed by dilutional RT-PCR. In contrast, the mRNA and protein for IL-13 was unaffected by FK506 in these same cultures. However, on further analysis we found that the secretion of IL-13, when induced with anti-IgE alone, is, like IL-4, inhibited by FK506. Thus, these results would suggest that there is an IL-3-dependent pathway for IL-13 generation in basophils that is resistant to FK506 and that is distinct from that induced through IgE-dependent signaling. In fact, the IL-13 protein induced by IL-3 alone was also unaffected by this drug. Although little is known with respect to signaling through the IL-3 receptor, there is evidence to suggest the involvement of JAK kinases that, in turn, activate STAT transcription factors (40, 41). If so, then this may explain why FK506 did not inhibit IL-3-induced IL-13 secretion, since this drug is not known to affect this pathway. It is important, however, to note that the translocation of NF-AT into the nucleus has been shown to be calcium dependent and is regulated by the calcium-dependent protein phosphatase, calcineurin. Both FK506 and cyclosporin A exert their inhibitory effects on cytokine transcription by preventing the dephosphorylation of calcium-dependent calcineurin, thereby preventing the translocation of NF-AT into the nucleus (42). In this study, FK506 was a potent inhibitor of IgE-dependent IL-4 and IL-13 production from basophils, supporting the concept that a calcineurin/NF-AT pathway not only is important for IL-4 activation, but probably has a role in IL-13 expression. Within this context, we observed that ionomycin-induced secretion of both IL-4 and IL-13, the most potent stimulus for either cytokine, is also ablated with FK506.
As noted above, PMA was a potent inducer of IL-13 secretion from
basophils. When the effect of FK506 on PMA-stimulated IL-13 secretion
was examined, we observed no inhibitory effect. Thus, the PKC-specific
pathway that results in IL-13 secretion appears to operate through a
transcription factor unrelated to NF-AT and may very well include a
pathway involving NF-B. This hypothesis is particularly intriguing
given the evidence noted above regarding the observations that PMA has
a negative effect on IL-4 generation by basophils and that NF-B
competes with NF-AT for binding sites within the IL-4 promoter. Thus,
it is possible that signal transduction mechanisms resulting in the
activation of IL-13 may, in fact, result in the down-regulation of
IL-4. This may also partially explain why IL-13 generation begins at a
time (2–4 h) when IL-4 secretion is nearly complete. Unlike FK506, the
PKC inhibitor BIS II did inhibit PMA-induced IL-13 secretion. However,
this drug, when used at the same concentration (1 µM), was found to
have a slight enhancing effect on the IL-13 induced by either IL-3 or
anti-IgE, suggesting that neither of these stimuli is involved in a
PKC-dependent pathway for IL-13 generation. In this instance, the
physiologic stimuli regulating the signals that modify PKC activity and
the specific isozymes of PKC involved have not yet been identified.
In conclusion, we have evidence showing that IL-4 secretion and IL-13 secretion from basophils differ in terms of their kinetics of secretion, their response to IL-3 and PMA stimulation, and their pharmacologic control. IL-4 and IL-13 are important mediators in the so-called Th2-type immune reactions and, hence, the cell source of these cytokines and the mechanisms involved in their secretion are of considerable interest. A greater understanding of the intracellular signals controlling IL-4 and IL-13 production may enable the development of therapeutic agents that specifically control the generation of these cytokines.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John T. Schroeder, Division of Clinical Immunology, Unit Office 2, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 22124.
3 Abbreviations used in this paper: BIS II, bisindolylmaleimide II; IMDM, Iscove’s modified Dulbecco’s medium; PAG, piperazine-N,N'-bis(2-ethane sulfonic acid)/albumin/glucose; C-IMDM, conditioned Iscove’s modified Dulbecco’s medium; HPRT, hypoxanthine phosphoribosyl transferase; PKC, protein kinase C; NF-AT, nuclear factor-AT.
Received for publication June 4, 1997. Accepted for publication October 31, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B: implications for differential gene expression in T helper cell subsets. Proc. Natl. Acad. Sci. USA 92:11623.RI-mediated induction of nuclear factor of activated T-cells. J. Biol. Chem. 270:16333.This article has been cited by other articles:
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  J. T. Schroeder, K. L. Chichester, and A. P. Bieneman Human Basophils Secrete IL-3: Evidence of Autocrine Priming for Phenotypic and Functional Responses in Allergic Disease J. Immunol., February 15, 2009; 182(4): 2432 - 2438. [Abstract] [Full Text] [PDF]
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P. Schmid-Grendelmeier, F. Altznauer, B. Fischer, C. Bizer, A. Straumann, G. Menz, K. Blaser, B. Wuthrich, and H.-U. Simon Eosinophils Express Functional IL-13 in Eosinophilic Inflammatory Diseases J. Immunol., July 15, 2002; 169(2): 1021 - 1027. [Abstract] [Full Text] [PDF]
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 G.-J. BRAUNSTAHL, S. E. OVERBEEK, W. J. FOKKENS, A. KLEINJAN, A. R. MCEUEN, A. F. WALLS, H. C. HOOGSTEDEN, and J.-B. PRINS Segmental Bronchoprovocation in Allergic Rhinitis Patients Affects Mast Cell and Basophil Numbers in Nasal and Bronchial Mucosa Am. J. Respir. Crit. Care Med., September 1, 2001; 164(5): 858 - 865. [Abstract] [Full Text] [PDF]
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F. H. Falcone, H. Haas, and B. F. Gibbs The human basophil: a new appreciation of its role in immune responses Blood, December 15, 2000; 96(13): 4028 - 4038. [Full Text] [PDF]
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 A. Oriente, N. S. Fedarko, S. E. Pacocha, S.-K. Huang, L. M. Lichtenstein, and D. M. Essayan Interleukin-13 Modulates Collagen Homeostasis in Human Skin and Keloid Fibroblasts J. Pharmacol. Exp. Ther., March 1, 2000; 292(3): 988 - 994. [Abstract] [Full Text]
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C. A. Oskeritzian, Z. Wang, J. P. Kochan, M. Grimes, Z. Du, H.-W. Chang, S. Grant, and L. B. Schwartz Recombinant Human (rh)IL-4-Mediated Apoptosis and Recombinant Human IL-6-Mediated Protection of Recombinant Human Stem Cell Factor-Dependent Human Mast Cells Derived from Cord Blood Mononuclear Cell Progenitors J. Immunol., November 1, 1999; 163(9): 5105 - 5115. [Abstract] [Full Text] [PDF]
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 T. R. Johnson and B. S. Graham Secreted Respiratory Syncytial Virus G Glycoprotein Induces Interleukin-5 (IL-5), IL-13, and Eosinophilia by an IL-4-Independent Mechanism J. Virol., October 1, 1999; 73(10): 8485 - 8495. [Abstract] [Full Text] [PDF]
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K. Miura, J. T. Schroeder, W. C. Hubbard, and D. W. MacGlashan Jr. Extracellular Signal-Regulated Kinases Regulate Leukotriene C4 Generation, But Not Histamine Release or IL-4 Production from Human Basophils J. Immunol., April 1, 1999; 162(7): 4198 - 4206. [Abstract] [Full Text] [PDF]
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Y. Li, F. E. R. Simons, and K. T. HayGlass Environmental Antigen-Induced IL-13 Responses Are Elevated Among Subjects with Allergic Rhinitis, Are Independent of IL-4, and Are Inhibited by Endogenous IFN-{gamma} Synthesis J. Immunol., December 15, 1998; 161(12): 7007 - 7014. [Abstract] [Full Text] [PDF]
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