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RI Signaling in Syk-Deficient Nonreleaser Basophils and IL-3-Induced Recovery of Syk Expression and Secretion1
Department of Pathology, University of New Mexico, Albuquerque, NM 87131
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, by releasing histamine and other
mediators from granules, producing IL-4 and other cytokines and, as
shown in this study, by forming membrane ruffles and showing increased
very late Ag-4 (VLA-4)-mediated adhesion to VCAM-1-expressing target
cells. We have identified five blood donors whose basophils lack
detectable levels of the FcRI-associated protein tyrosine kinase,
Syk. Despite showing no obvious ultrastructural differences from normal
basophils, nonreleaser basophils fail to form membrane ruffles, to show
increased VLA-4-mediated adhesive activity, or to produce IL-4 in
response to FcRI cross-linking. Although Syk protein levels are
suppressed in basophils from all five donors, Syk mRNA is consistently
present. Furthermore, culturing nonreleaser basophils for 4 days with
IL-3 restores Syk protein expression and FcRI-mediated histamine
release. Understanding the reversible suppression of Syk protein
expression in nonreleaser basophils, and learning to replicate this
property in patients with allergic inflammation could be a powerful and
specific way to limit symptomatic disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, and
respond to cross-linking of this receptor with functional responses
that include the secretion of inflammatory mediators, the production of
IL-4, IL-13, and other cytokines, and other responses, including two
described for the first time in this study, membrane ruffling, and the
up-regulation of very late Ag-4
(VLA-4)4-mediated
adhesive activity. Some or all of these responses very likely
contribute to the recruitment of basophils to sites of allergic
reactions and to their participation in the pathogenesis of allergic
inflammation. In particular, basophils are strongly implicated in
the late phase of allergic asthma (1) and are particularly
prominent in the airways of people who died of asthma (C. L. Kepley and
M. F. Lipscomb, unpublished observations).
The strong correlation between basophil recruitment and degranulation
and allergic disease adds interest to reports that basophils from
10% of donors fail to release histamine in response to FcRI
cross-linking (2, 3, 4, 5, 6). Non-releaser basophils degranulate
in response to stimuli such as
N-formyl-methionyl-phenylalanine (fMet peptide),
Ca2+ ionophore, and PMA, suggesting that their
lack of Ag-induced secretion results from the failure of early events
specific to the FcRI signaling cascade (3, 4, 5, 6). Previous
investigators established that FcRI expression and subunit
composition are normal in nonreleaser basophils (3, 5). We
found that basophils from three nonreleaser donors lacked detectable
levels of the FcRI-associated protein tyrosine kinase, Syk
(6). Remarkably, Syk levels were normal in B cells,
eosinophils, and neutrophils from the same donors. From these results,
we hypothesized that a basophil-specific suppression of Syk protein
levels may contribute importantly to the nonreleaser phenotype.
In this study, we show that Syk-deficient, nonreleaser basophils from
five separate donors are ultrastructurally normal, but fail to ruffle,
produce cytokines, or up-regulate their adhesive properties in
response to FcRI cross-linking. We also show that nonreleaser
basophils contain Syk mRNA and that Syk protein expression and
FcRI-dependent secretion can be restored by incubation with
IL-3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human IgE was prepared from human myeloma plasma (Cortex
Biochem, San Leadra, CA). Anti-Syk and anti-Lyn Abs were from Santa
Cruz Biotechnology (Santa Cruz, CA). Affinity-purified goat
anti-human IgE Ab (anti-IgE) was from Biosource (Camarillo,
CA). The anti-CD49d mAb HP2/1, a blocking Ab to the human
4
1 integrin (VLA-4),
was from Immunotech (Marseilles, France). The calcium ionophores A23187
and ionomycin, fMet peptide, PMA, IL-3, RPMI 1640, Ham’s F-12, and
IMDM were from Sigma (St. Louis, MO). mAb 22E7 to the FcRI--chain
was a generous gift from Dr. J. Kochan, Hoffman-LaRoche (Nutley, NJ).
The stimulatory mAb 8A2 to human VLA-4 was a generous gift from Dr. J.
Harlan (University of Washington, Seattle, WA). Human
VCAM-1-transfected Chinese hamster ovary (CHO) cells (VCAM-CHO cells)
were kindly provided by Dr. D. Leavesley (Hanson Cancer Center,
Adelaide, Australia).
Isolation of peripheral blood cells
Basophils were obtained by Percoll gradient centrifugation of
venous blood from normal donors with no history of allergic symptoms,
as described previously (7, 8). Purities from this initial
step ranged from 15 to 66%. Basophil purity was routinely increased to
>95% by negative selection using a negative selection cocktail from
StemCell Technologies (Vancouver, British Columbia, Canada) and
MidiMacs (Miltenyi Biotec, Auburn, CA) magnetic columns (described in
Ref. 8). In many experiments, negative selection was
followed by flow sorting (7) to yield >99.9% pure
basophils. In the ultrastructural studies below, 0.5–1 x
105 RBC (obtained after the initial Percoll step)
were added back to make the basophil pellet large enough for convenient
embedding and sectioning.
In vitro culture of basophils
Negatively selected basophils (>95% pure) were cultured for 4 days at a concentration of 1–5 x 105 cells/ml in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml of streptomycin, 10 µg/ml of Fungizone (RPMI-FCS medium), with or without 20 U/ml IL-3. The proportion of viable cells on day 4 was 75% (range 55–81%) for the IL-3-treated cells and 56% (range 43–62%) for non-IL-3-treated cells, as determined by trypan blue exclusion. For functional assays, these cells were primed with 10 µg/ml human IgE during the final hour of IL-3 incubation. They were then harvested, and viable cells were counted and resuspended to the concentrations indicated in the various assays. For Western blotting and RT-PCR analyses, the cells were flow sorted to >99.9% purity before use.
Transmission electron microscopy (TEM)
Suspensions of Percoll-enriched, negatively selected IgE-primed basophils were incubated at 37°C in prewarmed HBSS+ (HBSS with 1 mM CaCl2 and 1 mM MgCl2) with or without anti-IgE (0.1 µg/ml) for 30 min. In some experiments, cells were incubated in IL-3 as above. Cells were collected by centrifugation, RBC added back as described above to increase pellet volume, and pellets were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 30 min at room temperature, rinsed with cacodylate, and processed, as described before (9). Thin sections were observed using a Hitachi 600 transmission electron microscope.
Histamine release
Suspensions of Percoll-enriched, negatively selected IgE-primed basophils were washed with HBSS- (HBSS without Ca2+ or Mg2+) and suspended to 0.5–1.1 x 106 basophils/ml, and 100-µl aliquots were incubated at 37°C in prewarmed HBSS+ containing no addition (spontaneous secretion) or with the addition of anti-IgE or other activating agents (stimulated secretion). Reactions were terminated by dilution in ice-cold PBS and centrifugation, and histamine in cell pellets and supernatants was measured using an RIA (Alpco, Windham, NH), as described (6, 8). Total histamine was measured in supernatants obtained by freeze thawing cell aliquots in PBS/EDTA, followed by centrifugation at 10,000 x g for 5 min to remove debris.
VLA-4 surface expression
Peripheral blood basophils were harvested from releaser and
nonreleaser donors by Percoll gradient centrifugation and negative
selection, as described above. Cells were incubated for 10 min at
37°C with or without the anti-FcRI mAb 22E7 (3 µg/ml). Cells
were then lightly fixed at room temperature for 5 min with 0.5%
paraformaldehyde, washed, and incubated with FITC-conjugated
anti-CD49d mAb (HP2/1) at 1:50 in PBS/BSA (0.2%) for 30 min at
room temperature. The same Ab at the same concentration was
simultaneously added to 50 µl of Quantum Simply Cellular Microbeads
(Flow Cytometry Standards, San Juan, PR) per the manufacturer’s
recommendations. Cells and beads were washed in PBS and resuspended for
FACS analysis. Mean fluorescence of cells was compared, and binding
sites were quantified in resting and 22E7-treated (activated) cells
relative to the fluorescence of the Quantum Simply Cellular Microbead
standards.
VLA-4-mediated adhesive activity
The adhesive activity of basophils was measured using a
modification of the assay of Leavesley et al. (10).
Percoll-enriched, negatively selected IgE-primed basophils (5 x
105 basophils/ml) were suspended in RPMI-FBS
medium and fluorescence labeled by incubation for 45 min with 4 µg/ml
dihydroethidium (Molecular Probes, Eugene, OR) in a 5%
CO2 incubator. In parallel, VCAM-CHO cells
(2 x 106 cells/ml) were suspended in 1 ml
of Ham’s F-12 Nutrient Mixture, 10% FBS, 200 mM
L-glutamine, penicillin-streptomycin, and 1% sodium
pyruvate (Ham’s FBS medium), and fluorescence labeled with 1.5 µM
Fluo-3 AM (Molecular Probes). Cells were washed once in the medium used
for fluorescence labeling and once in adhesion buffer (modified
HBSS+ with 0.9 mM calcium and 0.35 mM magnesium).
Each group of cells was then suspended in 400 µl adhesion buffer at
37°C. For adhesion assays, cells were combined at a ratio of 1:3.5
basophil:VCAM-CHO cells in adhesion buffer at 37°C to a final volume
of 400 µl, and duplicate samples were incubated on a rocker in the
presence of no addition, anti-IgE (1 µg/ml), or anti-FcRI
mAb 22E7 (3 µg/ml) and other activating agents. Every experiment
included samples with added EDTA that reduces VLA-4 to its least active
conformation, and with added mAb 8A2 plus Mn2+,
which brings VLA-4 to its maximally active conformation
(11). In some experiments, parental (nontransfected) CHO
cells were used as additional controls for VLA-4-independent adhesion.
After 15 min, cells were fixed by adding 400 µl of 2%
paraformaldehyde and continued rolling for 2 min, and the proportion of
dually fluorescent conjugates formed between basophils and VCAM-CHO
cells was measured in a FACScaliber flow cytometer. The percentage of
basophils forming conjugates is derived by dividing the number of
conjugates (dual color events) by the number of conjugates plus free
basophils.
IL-4 production
Percoll-enriched, negatively selected IgE-primed basophils (0.1–1 x 105 basophils in 100 µl of IMDM containing 5% heat-inactivated FBS, 1x nonessential amino acids, and 5 µg/ml gentamicin) were incubated for 4 h at 37°C in a 5% CO2 incubator with or without the addition of anti-IgE or other activating agents. Cells were centrifuged, and IL-4 protein was measured in the cell-free supernatants by ELISA (Biosource; sensitivity = 0.27 pg/ml).
Western blotting
Expression of Lyn and Syk was measured in highly purified basophils (>99.9% pure; freshly isolated or IL-3 treated for 4 days) by Western blotting, as previously described (6, 8).
RT-PCR
mRNA for Syk was measured in highly purified basophils (>99.9%
pure) by RT-PCR. Total cellular RNA was prepared with the RNAeasy total
RNA system (Qiagen, Hilden, Germany) from 1–5 x
105 basophils. The monocyte/lymphocyte cell
population (no basophils) obtained after the Percoll gradient
centrifugation was used as a positive control. RNA concentrations were
determined by a spectrophotometric 260/280 ratio. RT-PCR was performed
using the Titan RT-PCR one-reaction system from Boehringer
(Indianapolis, IN). The following primers for human Syk or -actin
were obtained from the Protein Chemistry Laboratory at University of
New Mexico: Syk, 5'-TCCGAGCCAGAGACAACAACGG-3' and
5'-TTCCAGCGTCAGCAGCTTTCG-3'; -actin,
5'-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3' and
5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3'. The first cDNA strand was
obtained using avian myeloblastoma leukemia virus at 50°C for 30 min,
and followed by PCR using the Expand High Fidelity enzyme blend. The
RT-PCR were done in a total volume of 30 µl containing 50 mM KCl, 10
mM Tris-HCl, pH 9, 1.5 mM MgCl2, 200 µM dNTPs,
and 10 pmol of each primer. PCR conditions were: template denaturation
(1 min at 95°C), followed by a 35-fold repetitive cycle of 1 min at
56°C (annealing), 2 min at 72°C (extension), and 1 min at 95°C
(denaturation). After amplification, the samples were analyzed by
electrophoresis on a 2% gel containing ethidium bromide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By TEM, unstimulated releaser (Fig. 1
A) and nonreleaser (Fig. 1C) basophils appear as rather typical granulocytes, with
multilobed nuclei, relatively sparse endoplasmic reticulum and
mitochondria, and fairly smooth plasma membranes. Granules are
numerous, and their content of loosely packed matrix material
plus occasional dense core material, multilamellar membranes, and
internal vesicles is not noticeably different between
releaser and nonreleaser cells.
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RI cross-linking induces degranulation and membrane ruffling
in releaser but not nonreleaser basophils
In releaser basophils, FcRI cross-linking induces the
intracellular fusion of granules and release of granule contents (Fig. 1
B), as previously described (12). Releaser
basophils also show a dramatic membrane ruffling response to FcRI
cross-linking. In contrast, granules in nonreleaser basophils remain
individual and intact following FcRI cross-linking (Fig. 1D). Furthermore, there is no membrane ruffling response
to FcRI cross-linking in the nonreleaser cells.
FcRI cross-linking up-regulates VLA-4-mediated adhesion in
releaser but not nonreleaser basophils
The adhesive activity of basophil VLA-4 toward VCAM-1 was
determined by separately labeling basophils and VCAM-CHO cells with
nonoverlapping fluorochromes, then mixing the cells with or without the
addition of activating agents, and measuring conjugate formation
between these distinct cell types during a rolling assay. The results
in Fig. 2A show there is
essentially no conjugate formation between releaser basophils and
VCAM-CHO cells when the cells are coincubated in the presence of
blocking Abs to VLA-4 (or VCAM-1; data not shown) and when EDTA is
included in the assay mixture to minimize the binding activity of
VLA-4. Under the conditions of our assay, 50% of releaser basophils
form conjugates with VCAM-CHO cells when the VLA-4-activating mAb, 8A2,
is included, with the VLA-4-activating cation,
Mn2+, in the incubation mixture. These data
establish the critical role of VLA-4 and its counterreceptor VCAM-1 in
conjugate formation and set the lower and upper limits of
VLA-4-mediated adhesion in our assay. Approximately 10–15% of
releaser basophils form conjugates with VCAM-CHO cells when the two
cell types are rolled together in adhesion buffer (HBSS with
Ca2+ and Mg2+) alone.
FcRI cross-linking by the addition of anti-IgE (shown) or
mAb 22E7 (not shown) during the adhesion assay increases the
proportion of releaser basophils forming conjugates with VCAM-CHO cells
to 25–40%. Conjugate formation between releaser basophils and
VCAM-CHO cells is also increased by incubation with fMet peptide that
activates the G protein-coupled chemoattractant receptor, with
ionomycin that mobilizes Ca2+, and with PMA whose
intracellular targets include protein kinase C.
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B recapitulate key controls for
nonreleaser basophils. In particular, the effects of EDTA in
down-regulating adhesive activity and of mAb 8A2 plus
Mn2+ in up-regulating adhesion are very similar
between releaser and nonreleaser basophils. Additionally, conjugate
formation between nonreleaser basophils and VCAM-CHO cells is increased
by incubation with fMet peptide, ionomycin, and PMA. However, the
proportion of unstimulated nonreleaser basophils forming conjugates
with VCAM-CHO cells during incubation in adhesion medium is lower than
the proportion of unstimulated releaser cells that form conjugates.
Furthermore, FcRI cross-linking causes no increase in nonreleaser
basophil adhesion to VCAM-CHO cells.
We explored the possibility that the low basal adhesiveness of
nonreleaser basophils could be attributed in part to low levels of
VLA-4 on the nonreleaser basophil surface. The results of
immunophenotyping studies showed that releaser and nonreleaser
basophils express very similar amounts of membrane VLA-4 (Fig. 2C).
FcRI-mediated IL-4 production is blocked in nonreleaser
basophils
The results in Fig. 3A
show that incubating releaser basophils with anti-IgE for 4 h
induces the production of IL-4. The optimal concentration of
anti-IgE for IL-4 production is 0.1 µg/ml, less than the optimal
anti-IgE concentration for secretion (see above). Releaser
basophils also produce IL-4 in response to stimulation with the
Ca2+ ionophore, A23187, but not with fMet peptide
(not shown). We were not able to detect IL-4 in nonreleaser basophil
cultures stimulated with optimal concentrations of anti-IgE for
4 h. Longer incubations (8 h) under the same conditions also did
not induce IL-4 secretion (data not shown). Nevertheless, A23187
induced IL-4 production in nonreleaser basophils.
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RI-mediated secretion is partially restored by incubating
nonreleaser basophils with IL-3
The results in Fig. 4A
confirm previous evidence (2, 3, 4, 5, 6) that freshly isolated
nonreleaser basophils show little or no secretion in response to
varying concentrations of anti-IgE, even though they degranulate
normally to A23187, fMet peptide, and PMA.
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B
that 4-day incubation of nonreleaser basophils in medium with 20 U/ml
of IL-3 induces a partial recovery of FcRI-mediated secretion. Fig. 4B also shows that releaser basophils cultured under the
same conditions continue to degranulate in response to anti-IgE.
Consistent with the previous report (5), long-term culture
with IL-3 resulted in an increased sensitivity to anti-IgE; the
optimal concentration of anti-IgE for secretion by releaser
basophils shifted from 1 µg/ml in freshly purified basophils (Fig. 4A) to 0.1 µg/ml after culture with IL-3 for 4 days (Fig. 4B).
IL-3 incubation does not restore all FcRI-mediated responses in
nonreleaser basophils. The results in Fig. 2D show that
incubation for 3 days with IL-3 induces a partial recovery of the
FcRI-stimulated up-regulation of VLA-4 adhesive activity of
nonreleaser basophils. The result was variable between donors, and
the recovery was not complete. Similarly, nonreleaser basophils
observed by TEM after incubation with IL-3 and activation with
anti-IgE showed consistent evidence of granule-granule fusion, but
inconsistent membrane-ruffling responses (data not shown). Not even a
partial recovery of anti-IgE-induced IL-4 production was observed
in nonreleaser basophils that were incubated for 4 days with IL-3 (Fig. 3B).
Nonreleaser basophils contain Syk mRNA and express Syk after incubation with IL-3
In our previous work (6), Syk protein was not detected by Western blotting in basophils from three nonreleaser donors. Western blotting of lysates of freshly isolated nonreleaser basophils from two additional donors identified since our initial publication also showed no Syk protein, except after very prolonged exposure of the gels (data not shown).
Although Syk protein was very difficult to detect, Syk mRNA was readily
detected in basophils from all five nonreleaser donors. Syk mRNA was
also detected in basophils from all releaser donors tested.
Representative data from these RT-PCR analyses are shown in Fig. 5A.
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B.
Syk protein was not detectable in nonreleaser basophils that were
cultured for 4 days without IL-3, then repurified by flow sorting for
Western blot analysis (Fig. 5B, lanes 3 and
6). In contrast, Syk protein expression was maintained in
releaser basophils (Fig. 5B, lanes 1 and
4) and was restored in nonreleaser basophils (Fig. 5B, lanes 2 and 5) when the culture
medium contained added IL-3. In six replicate experiments, Syk
expression in nonreleaser basophils was always restored by IL-3
incubation. Densitometric analysis using Syk-positive B/T cells as a
reference showed basophil Syk levels that were between 60 and 95% of
the initial Syk levels of lymphocytes from the same donor (data not
shown). Lyn was previously detected in freshly isolated releaser and
nonreleaser basophils (4). The results in Fig. 5, lanes 1–6, show additionally that Lyn is still expressed in
both releaser and nonreleaser basophils after 4 days of in vitro
incubation without and with IL-3. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI
cross-linking activates the protein tyrosine kinases Lyn, Syk, and
ZAP-70, resulting in the phosphorylation of multiple signaling proteins
and leading to basophil degranulation (8). However,
basophils from 10% of donors fail to release histamine in response
to FcRI cross-linking (2, 3, 4, 5, 6). Previous analyses in our
laboratory revealed that basophils from 3 of 37 (now 5 of 42)
donors showed no FcRI-dependent secretion and also lacked detectable
levels of the FcRI-associated protein tyrosine kinase, Syk
(6). Remarkably, Syk levels were normal in B cells,
eosinophils, and neutrophils from the same donors. From these results,
we hypothesized that a basophil-specific loss of Syk may underlie the
nonreleaser phenotype.
In this study, we show that the ultrastructure of nonreleaser basophils
is indistinguishable from that of resting releaser basophils, reducing
the likelihood that the nonreleaser phenotype results from basophil
immaturity or abnormal granule morphology, and we confirm by direct
morphological observation that only releaser basophils respond to
FcRI cross-linking by degranulation.
Other functional defects have not been vigorously explored in
nonreleaser basophils. We report that nonreleaser basophils have
multiple functional defects in FcRI signaling in addition to
impaired degranulation.
First, releaser basophils form membrane ruffles in response to FcRI
cross-linking. Ruffling is a well-known response of RBL-2H3 mast cells
to FcRI cross-linking and has been implicated in the stimulation of
macropinocytosis that also occurs when these cells are activated
(9, 13). In contrast to releaser basophils, nonreleaser
basophils do not form membrane ruffles in response to FcRI
cross-linking.
Second, releaser basophils up-regulate their VLA-4-mediated adhesion to
VCAM-1-transfected CHO cells in response to FcRI cross-linking. The
modulation of basophil VLA-4 activity by signals from the FcRI
signaling pathway is predictable based on a substantial literature
linking tyrosine kinase-dependent signaling pathways in T lymphocytes
and many other cells to the activation of integrins (14, 15). Basophil adherence to endothelium is mediated in part by
interactions between VLA-4 on the basophils and VCAM-1 on the
endothelial cells (16). Thus, the up-regulation of VLA-4
adhesive activity in activated basophils very likely stimulates the
recruitment of cells from blood to inflamed tissues. Nonreleaser
basophils fail to up-regulate VLA-4-mediated adhesion in response to
FcRI cross-linking. In contrast, fMet peptide, ionomycin, and PMA,
which signal from the cytoplasm to the integrin, and mAb 8A2 plus
Mn2+, which signal from the medium to the
integrin, all enhance the VLA-4-mediated adhesion of nonreleaser
basophils to VCAM-CHO cells. These results localize the adhesion
deficit to defective FcRI signaling and not to impaired VLA-4
responsiveness.
Third, releaser basophils produce IL-4 in response to FcRI
cross-linking and also in response to treatment with
Ca2+ ionophores. Previous investigators have
implicated basophil-derived IL-4 in the stimulation of allergic
inflammation (17). We report that nonreleaser basophils
are unable to produce IL-4 in response to anti-IgE stimulation.
Again the defect is at the level of signal initiation, because IL-4
production in response to A23187 is normal.
Non-releaser basophils have been observed to recover FcRI-mediated
secretory activity after incubation for 3 days or longer in IL-3
(5). We confirmed that incubating nonreleaser basophils
for 4 days in medium with IL-3 indeed causes a substantial recovery of
FcRI-mediated histamine release. Importantly, the restoration of
secretion following 4 days of incubation with IL-3 was accompanied by
the restoration of Syk protein expression in nonreleaser basophils.
In additional experiments, we discovered, remarkably, that basophils from all five of our nonreleaser donors contain Syk mRNA. Thus, the very low levels of Syk protein in freshly isolated nonreleaser basophils clearly reflect a lineage-specific posttranscriptional abnormality in these cells. There is precedent in lymphocytes for regulated Syk protein expression, although the level of the regulation (pre- or posttranscriptional) has not been reported. In T cells, Syk levels vary as a function of both development and differentiation, with highest levels being found in thymocytes during the pre-TCR signaling stage, and lowest levels occurring in peripheral T cells (18, 19, 20). Additionally, a population of mIg-positive B chronic lymphocytic leukemia cells has been described that does not proliferate in response to BCR cross-linking, but does proliferate in response to signals that bypass the BCR. The resistance of these cells to Ag-induced proliferation was linked to their low levels of Syk expression in comparison with Ag-sensitive B chronic lymphocytic leukemia cells (21).
One explanation for the lack of Syk protein in vivo in nonreleaser basophils is insufficient circulating levels of IL-3 or other cytokines that provide signals for the basophil-specific translation of Syk mRNA to protein. This hypothesis is suggested by the recovery of Syk expression by in vitro incubation with IL-3. Variations in levels of IL-3 or other circulating cytokines could explain our earlier discovery that basophils from one nonreleaser donor were able to cycle between the releaser and nonreleaser phenotype based on both secretory activity and Syk protein expression (6). The difficulty of detecting IL-3 in serum from either releaser (n = 5) or nonreleaser (n = 3) donors using ELISA (sensitivity = <1 pg/ml; data not shown) has to date confounded attempts to test this hypothesis.
It is equally possible that Syk mRNA is translated in nonreleaser basophils to protein that is degraded too rapidly for detection by conventional Western blotting. In particular, the adaptor protein, Cbl, has been identified as a binding partner and negative regulator of Syk that reduces both Syk phosphorylation and Syk levels (22, 23). Because the RING finger domain of Cbl has ubiquitin-protein ligase activity (24), Cbl overexpression or excessive catalytic activity could promote the degradation of Syk protein in nonreleaser basophils by a proteasome-dependent mechanism. Additionally, it was recently reported that ZAP-70 is rapidly degraded in activated T cells by a calpain-dependent mechanism (25); excess calpain activity could equally well promote the rapid degradation of Syk protein in nonreleaser basophils by a proteasome-independent mechanism.
The possibility that nonreleaser basophils have additional signaling
defects downstream of Syk is being investigated. Lyn is consistently
present in these basophils. Nevertheless, the partial recovery of
FcRI-mediated VLA-4-mediated adhesion and no recovery of
FcRI-mediated IL-4 production suggest a possible dysregulation of
other signaling molecules.
It is increasingly clear that basophils are not just a surrogate for
the elusive mast cell for investigators studying allergic inflammation
in humans. Rather, Ag-stimulated basophils release mediators that
induce both acute and late phase allergic responses; they are major
sources of IL-4 and IL-13, key cytokines in the propagation of allergic
inflammation, and there is a strong relationship between clinical
improvement induced by anti-IgE immunotherapy and reduced
FcRI-mediated basophil degranulation (17, 26, 27). In
rodent models, all methods to eliminate Syk, use of Syk-selective
inhibitors (28, 29), of Syk-negative cell lines
(30), of cells from Syk knockout mice (31),
and by use of Syk antisense oligonucleotides (32) have
consistently eliminated most or all FcRI signaling responses.
However, human allergy is unlikely to be easily treated with Syk
inhibitors because this kinase is essential for many other functions,
including the development and function of T cells, B cells, and
platelets (20, 33). The nonreleaser basophil is clearly a
unique tool to understand how to reversibly suppress Syk expression in
human basophils without inhibiting its expression in other hemopoietic
cells. Treatments that replicate this property could help to protect
allergy sufferers against symptomatic disease.
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Acknowledgments |
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Footnotes |
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2 C.L.K. and L.Y. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Christopher L. Kepley, Department of Pathology, University of New Mexico School of Medicine, CRF Building, Room 203A, 2325 Camino de Salud, Albuquerque, NM 87131.
4 Abbreviations used in this paper: VLA-4, very late Ag-4; fMet, N-formyl-methionyl-phenylalanine; CHO, Chinese hamster ovary; TEM, transmission electron microscopy.
Received for publication April 7, 2000. Accepted for publication August 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-activated tyrosine kinases Lyn, Syk, and Zap-70 in human basophils. J. Allergy Clin. Immunol. 102:304.[Medline]
1 integrin (CD29) stimulates VLA-dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components. J. Cell Biol. 116:499.R1 cross-linking to increased cell substrate adhesion in RBL-2H3 tumor mast cells. J. Immunol. 152:270.[Abstract]
RI- surface densities and mediator release by anti-IgE infusions is reversible in vitro and in vivo. J. Immunol. 162:5624.R1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J. Biol. Chem. 269:29697. receptor I-mediated activation of Syk. Proc. Natl. Acad. Sci. USA 94:12539.This article has been cited by other articles:
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  D. W. MacGlashan Jr., S. Ishmael, S. M. MacDonald, J. M. Langdon, J. P. Arm, and D. E. Sloane Induced Loss of Syk in Human Basophils by Non-IgE-Dependent Stimuli J. Immunol., March 15, 2008; 180(6): 4208 - 4217. [Abstract] [Full Text] [PDF]
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J. J. Ryan, H. R. Bateman, A. Stover, G. Gomez, S. K. Norton, W. Zhao, L. B. Schwartz, R. Lenk, and C. L. Kepley Fullerene Nanomaterials Inhibit the Allergic Response J. Immunol., July 1, 2007; 179(1): 665 - 672. [Abstract] [Full Text] [PDF]
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L. A. Youssef, M. Schuyler, L. Gilmartin, G. Pickett, J. D. J. Bard, C. A. Tarleton, T. Archibeque, C. Qualls, B. S. Wilson, and J. M. Oliver Histamine Release from the Basophils of Control and Asthmatic Subjects and a Comparison of Gene Expression between "Releaser" and "Nonreleaser" Basophils J. Immunol., April 1, 2007; 178(7): 4584 - 4594. [Abstract] [Full Text] [PDF]
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