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Meakins-Christie Laboratories, Departments of Medicine and
Pediatrics, McGill University, Montreal, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and -2 subunits in human
peripheral blood eosinophils. Surface expression of IL-12R1 and
-2 subunits on freshly isolated human eosinophils was optimally
expressed after incubation with PMA. To determine the functional
significance of IL-12R studies, we studied cell viability and
apoptosis. Morphological analysis and propidium iodide staining for
cell cycle demonstrated that recombinant human IL-12 increased in vitro
human eosinophil apoptosis in a dose-dependent manner. Addition of IL-5
together with IL-12 abrogated eosinophil apoptosis, suggesting that
IL-12 and IL-5 have antagonistic effects. Our findings provide evidence
for a novel role for IL-12 in regulating eosinophil function by
increasing eosinophil apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue eosinophilia within the lungs and nose of individuals with allergic asthma and rhinitis has primarily been attributed to the influx of mature cells from the blood. However, increased eosinophil survival and/or decreased eosinophil apoptosis (programmed cell death) has also been recently proposed as a mechanism underlying tissue eosinophilia (6, 7). It is evident that Th2-type cytokines such as IL-4, IL-5, and GM-CSF are important mediators for eosinophil chemoattraction and survival in allergic inflammation (8, 9, 10). Recent studies have focused on the mechanisms involved in Th2-type cytokine-eosinophil interaction.
To mitigate against the effect of Th2 cytokines, strategies have been developed to suppressed their production, thus decreasing allergic inflammation. Glucocorticoids are the most useful class of drugs for treating many eosinophil-related disorders including allergic inflammation. Glucocorticoids can cause a striking reduction in eosinophil numbers in vivo (11). They exert inhibitory effects on eosinophil survival directly through specific receptors on eosinophils, as well as indirectly through inhibition of cytokines that promote eosinophil survival, such as IL-5 and GM-CSF (12).
A second strategy focuses on cytokines that have immunoregulatory effects, such as IL-12. Produced by APC (13), IL-12 induces Th1 cell proliferation and Th1-type cytokine expression (13, 14, 15, 16). IL-12 has also been proposed as an antiallergic cytokine by virtue of its inhibitory effects on Th2-type cytokine expression as well as eosinophilic inflammation (17, 18, 19, 20, 21). We have recently shown that the level of IL-12 mRNA expression in the airways of asthmatic subjects is significantly lower than that in nonasthmatic controls and was significantly increased in response to steroid treatment (22). It is unknown whether IL-12 can have a direct effect on eosinophil function.
There are two subunits of the IL-12R, IL-12R1 and IL-12R2
(23, 24), which belong to the gp130 group of the cytokine
receptor superfamily. Both subunits can bind IL-12 independently of one
another; however, interaction between them is required for IL-12
signaling. Furthermore, the IL-12R2 subunit acts as a high affinity
converter. Functional IL-12R have been recently described on human and
murine T cells (25, 26, 27) and on NK cells (28).
To date, IL-12R expression on eosinophils has not been determined. To
investigate the role of IL-12 on eosinophils and to understand the
mechanism by which IL-12 may exert its influence in the allergic
response, we examined the expression of IL-12R on eosinophils as well
as the effects of IL-12 on eosinophil survival.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rIL-12, rIL-5, rIFN-
, and rat IgG2a isotype control Ab were
purchased from R&D (Minneaopolis, MN). Anti-CD16 and anti-CD3 mAb
microbeads were from Miltenyl Biotec (Bergisch Gladbach, Germany). Rat
anti-human IL-12R1 and 2 IgG2a isotype Abs were gifts from
Dr. D. Presky, Hoffman LaRoche, Italy. Propidium iodide
(PI)3
was from Cedarlane (Toronto, Ontario, Canada). Biotinylated rabbit
anti-rat IgG was obtained from Vector Laboratories (Burlingame,
CA). FITC, Fast Red, and streptavidin-conjugated alkaline phosphatase
were purchased from Dako (Glostrup, Denmark). FCS was purchased from
HyClone Laboratories (Logan, UT). Dextran T70, LM-2 emulsion, and
35S were obtained from Pharmacia (Uppsala,
Sweden). Triton X, formamide, and RNase A, were obtained from Sigma
(St. Louis, MO).
Peripheral blood eosinophil purification
After informed consent, 50 ml blood were obtained from volunteers with peripheral blood eosinophilia ranging from 5 to 10% (n = 25). Human blood leukocytes were obtained from whole blood by sedimentation of RBC in 6% dextran T70. Granulocytes were enriched on a Ficoll-Hypaque gradient (1.077 g/ml; Pharmacia), and eosinophils were negatively selected from the enriched cells by immunomagnetic selection on a MACS column using anti-CD16 microbeads. Eosinophil purity was consistently >98%, with neutrophils being the only contaminating cells. The viability of freshly isolated eosinophils were >99% as determined by trypan blue dye exclusion.
Cell culture
Isolated human peripheral blood eosinophils (1 x
106/ml) were resuspended in RPMI 1640
supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml
penicillin, and 100 µg/ml streptomycin. Cells were cultured at 37°C
in a humidified atmosphere with IL-12 (10 ng/ml) in the absence or
presence of IL-5 (2.5 ng/ml) for 18 h. To investigate the
regulation of IL-12R expression, eosinophils were cultured in the
presence of IL-5 (2.5 ng/ml), IFN- (10 ng/ml), IL-12 (10 ng/ml),
IL-18 (10 ng/ml), IL-12 plus IL-18, PMA (1–10 ng/ml), or PMA plus
IL-12 plus IL-18 for 12 h.
Cytospin preparations
Cytospin slides were prepared from eosinophils freshly isolated from human peripheral blood or from cultured eosinophils. The cytospins were then fixed in 4% paraformaldehyde for 20 min at room temperature and washed with 0.05 M Tris-HCl-buffered isotonic saline (TBS), pH 7.6. After drying, the slides were stored at -20°C before immunocytochemistry, in situ hybridization, or morphological analysis of apoptosis.
Preparation of riboprobes
cDNA probes coding for IL-12R1 and -2 were inserted into
Bluescript vectors. The vectors containing either IL-12R1 or -2
cDNAs were linearized with the appropriate enzymes and transcribed in
vitro with either SP6 or T7 polymerases in the presence of
[35S]UTP to generate sense and antisense
riboprobes.
In situ hybridization
In situ hybridization was performed as previously described (29). Briefly, cytospin preparations were first permeabilized by immersion in 0.3% Triton X-100 in PBS for 10 min followed by exposure to proteinase K (1 mg/ml in 20 mM Tris-HCl and 1 mM EDTA, pH 7.2) for 30 min at 37°C. The slides were then prehybridized with 50% formamide in 2x SSC for 15 min at 37°C. Hybridization was performed with [35S]UTP-labeled riboprobes (either antisense or sense) for 16 h at 42°C. Posthybridization washings were done with SSC solution (4x SSC and 0.1x SSC) followed by RNase A treatment to remove unhybridized single-stranded RNA. The preparations were dehydrated, immersed in Amersham LM-2 emulsion, and then subjected to autoradiography for 18 days. The autoradiograms were developed in Kodak D-19 developer, fixed, and subsequently counterstained with hematoxylin. As the negative control, sections were hybridized with the sense probe or were pretreated with the RNase A before hybridization with the antisense probe.
Immunocytochemistry
IL-12R immunoreactivity was detected by the avidin-biotin
complex method, as previously described (30). Briefly, the
cytospin preparations of eosinophils were washed with TBS. After
saturation for 20 min with TBS containing 10% normal human serum and
5% normal goat serum (Cedarlane, Hornby, Ontario, Canada), cells were
incubated with rat anti-human IL-12R1 (BM10) or 2 (LM5) mAbs,
(each at 1/20 dilution) in Ab dilution buffer (Dako) overnight at
4°C. After being washed, the cells were incubated with biotinylated
rabbit anti-rat Ab for 30 min at room temperature followed
by streptavidin-conjugated alkaline phosphatase (1/100 dilution) for
1 h at room temperature. The slides were then developed using Fast
Red, counterstained with Mayer’s hematoxylin, and examined by
microscopy.
Flow cytometric analysis of IL-12R expression on eosinophils
Cells (2 x 105) were incubated with
saturating concentrations of primary Abs, either rat anti-human
IL-12R1, 2 mAbs or IgG2a isotype control Ab (1/100 dilution) in
PBS for 30 min at 4°C. The cells were washed twice with PBS and
incubated at room temperature for 30 min with biotinylated rabbit
anti-rat Ab (1/100 dilution). After being washed, the cells were
incubated with PE-conjugated streptavidin (1/100 dilution; Sigma, St.
Louis, MO) at room temperature for 30 min. Cells were washed and
resuspended in 200 µl PBS and analyzed on FACSCalibur flow cytometer
(BD Biosciences, Oxnard, CA). IL-12R expression was read using FL-2
channel and compared with the control.
Determination of eosinophil apoptosis by morphological assessment of nuclei alteration
Apoptosis was evaluated by morphological assessment (31). Briefly, cytospin preparations were stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA) to reveal nuclear morphology. Apoptotic cells were discriminated from healthy cells by the condensed and rounded appearance of their nuclei under light microscopy. Cells exhibiting apoptotic nuclei were enumerated in different fields in a blinded manner using a random coded order. A minimum of 500 total cells per slide was counted. Cells were then photographed under an Axioscope microscope (Zeiss, Oberkochen, Germany) at x400 magnification.
Cell cycle analysis
Apoptosis was assessed by cell cycle analysis using PI staining as described previously (32). Briefly, harvested eosinophils (0.2 x 106 cells) were washed twice with PBS by centrifugation at 1200 rpm for 10 min. The cells were then resuspended in 250 µl FCS and 250 µl RPMI 1640 and fixed in 1.5 ml 70% ethanol at 4°C for a minimum of 30 min. The cells were washed twice with PBS by centrifugation at 4°C and 2400 rpm for 5 min, resuspended in 1 ml of PBS with 0.05 mg/ml RNase A (50 U/mg), and incubated for 30 min at 37°C. After this, 50 µg/ml PI was added. Using flow cytometry (Cell Fit Software; BD Biosciences), cell cycle analysis was performed on eosinophils gated on the basis of their cell volume (right angle scatter) and cellular DNA content with any cell debris excluded from analysis. The percentage of apoptotic cells was determined in the hypodiploid peak.
Determination of eosinophil viability
The percentage of necrotic eosinophils was determined by using trypan blue exclusion. Spontaneous uptake of trypan blue as a vital dye by eosinophils was then assessed by light microscopy.
Quantification and statistics
In situ hybridization and immunocytochemistry slides were analyzed for positive signals in a blinded fashion by two independent examiners. Data are represented within the text and figures as the mean percent ± SEM. Comparisons between groups were performed using a one-way ANOVA and Fisher post hoc test. A value of p < 0.05 was considered statistically significant (SyStat version 7.1; SyStat, Evanston, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ hybridization was performed to examine the potential of
eosinophils to produce and express IL-12R. Hybridization of the
[35S]UTP-labeled antisense riboprobes and mRNA
encoding IL-12R1 and -2 (Fig. 1
, A and B) were demonstrated by specific deposits
of silver grains in the photographic emulsion overlying the cytospins.
IL-12R1 and -2 mRNA were expressed in eosinophils obtained from
all donors. The mean percentage of eosinophils expressing IL-12 R1
mRNA was 56.4 ± 6.6%, and that for IL-12R2 mRNA was 49.1
± 3.9 (Fig. 1C, mean ± SEM, n = 12).
The percentage of eosinophils expressing the IL-12R1 and -2 were
significantly higher in healthy controls than in asthmatics (mean
± SEM, IL-12R1: asthmatics, 36.1 ± 2.4; controls; 69.2% ±
7.01, p 
0.001. IL-12R2: asthmatics, 38.6 ±
4.4; controls; 59.5 ± 1.3, p 0.001). No
positive hybridization signals were observed when the sense probe was
used or when the cell preparations were pretreated with RNase before
hybridization with the antisense probe.
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Eosinophils exhibited positive immunoreactivity for IL-12R1 and
-2 as detected by the presence of discrete red cytoplasmic or
membranous staining (Fig. 2, A and B). Eosinophils obtained from all donors
were immunoreactive for IL-12R1 and -2 subunits. The mean
percentages of eosinophil immunoreactivity for IL-12R1 and -2
were 39.9 ± 4.8 and 65.9 ± 3.4, respectively (Fig.
2D). There was no difference between the percentages of
the IL-12R1 and -2 immunoreactivity between asthmatics and the
control subjects (data not shown).
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To differentiate surface from cytoplasmic expression of
IL-12R, freshly isolated peripheral blood eosinophils were
stained forsurface expression of IL-12R1 and -2 subunits
and analyzed by indirect immunofluorescence and flow cytometry.
Eosinophils were obtained from a total of eight donors. In four of the
eight eosinophil preparations, IL-12R1 and -2 were detected on
eosinophil cell surface (Fig. 3, A and B). Of these four positive preparations,
three were from asthmatic patients and one was from a nonasthmatic
healthy control. The percentages of eosinophils expressing IL-12R1
and IL-12R2 were 27.3 ± 3.6 and 14.3 ± 1.1,
respectively, in the four subjects studied (Fig. 4). Baseline surface staining for IL-12R was not detectable in four
subjects (two asthmatics and two nonasthmatic healthy controls) of
eight donors (Fig. 3, C and D). Although we were
unable to detect IL-12R expression by FACS analysis, using
immunocytochemistry technique and the same Abs, we demonstrated the
IL-12R1 and -2 expression in eosinophils from these four donors
(data not shown).
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PMA (1–10 ng/ml) stimulation increased IL-12R1 and -2
surface expression on eosinophils (IL-12R1, 48.4 ± 4.5;
IL-12R2, 49.8 ± 12.8) compared with unstimulated eosinophils
(Figs. 3
and 4). In addition, PMA stimulation induced IL-12R surface
expression by FACS analysis on eosinophils with undetectable baseline
IL-12R expression (Fig. 3, C and D). Eosinophils
cultured with rIL-5 (2.5 ng/ml), rIL-12 (10 ng/ml), rIL-18 (10 ng/ml),
or rIFN- (10 ng/ml), did not exhibit an increase in IL-12R
expression.
Effect of IL-12 on eosinophil apoptosis
In the light of previous data that demonstrate decreased
eosinophilia on exogenous administration of IL-12 and the presence of
IL-12R on eosinophils, we examined the effect of IL-12 on the
maintenance of eosinophil survival. Eosinophils were cultured with
rIL-12, rIL-5, or medium alone for 18 h, and the extent of
apoptosis was evaluated by morphological analysis (27)
(Fig. 5A). Fig. 5B
demonstrates that IL-12 significantly increased the percentage of
eosinophils undergoing apoptosis (24.3 ± 3.8, mean percent of
apoptotic cells ± SEM) compared with IL-5 (8.8 ± 2.0%,
p < 0.005) and medium control (13.2 ± 2.7%,
p < 0.05, n = 5). The effect of IL-12
on eosinophil apoptosis was dose dependent, with apoptosis increasing
at IL-12 concentrations ranging from 1 to 100 ng/ml (Fig. 6).
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, A–D). After 18 h of cell culture with IL-12,
eosinophil apoptosis was 2-fold higher than that of IL-5 (Fig. 7D, IL-12 vs IL-5; 24.3 ± 2.9% vs 12.7 ± 3.5%,
mean percent of apoptotic cells ± SEM, p < 0.05,
n = 4). Staining with trypan blue demonstrated that
there was no difference in the percentage of necrotic cells detected
between different conditions (Fig. 7E).
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IL-5 serves as a survival factor for eosinophils. When mature
eosinophils are cultured in the absence of IL-5, they undergo apoptosis
(8, 9, 10). The effect of IL-12 on eosinophil apoptosis was
further investigated in the presence of IL-5. Eosinophils were cultured
with rIL-5 (2.5 ng/ml) and rIL-12 (10 ng/ml) for 18 h, and cell
morphology was examined. In the absence of IL-5, IL-12 significantly
increased the number of eosinophils undergoing apoptosis, compared with
IL-5 or medium alone (Fig. 8). The percentage of apoptotic eosinophils in the presence of IL-12 was
30.8 ± 8.4 (n = 3), whereas it was 17.1 ± 4
in IL-5 (IL-12 vs IL-5; p 0.01) and 21.4 ± 3
in medium alone (IL-12 vs medium; p 0.05) (Fig. 8B). IL-5 rescued eosinophils from programmed cell death
induced by IL-12 (Fig. 8B, IL-12 plus IL-5 vs IL-12;
16.1 ± 3.1% vs 30.8 ± 8.4%, p < 0.05,
n = 3).
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demonstrates that after a 3-h incubation in IL-12, IL-5 was no longer
able to reverse the effect on eosinophil apoptosis. Conversely,
incubation with IL-5 for 3 h or more rendered the eosinophils
insensitive to the action of IL-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and -2 subunits (
Figs. 1–4).
This is based on direct demonstration of mRNA and protein, as well as
evidence that IL-12 accelerates eosinophil apoptosis (
Figs. 5–7),
which may be reversed by addition of IL-5 (Fig. 8).
IL-12 receptors consist of two subunits, 1 and 2. The
IL-12R1 is ubiquitously expressed, yet its role in IL-12 signaling
is still unknown. IL-12R2 is the signal-transducing unit and has a
more limited expression, to date being detected in T and B cells and NK
cells (25, 28, 33). Our data demonstrating the presence of
both receptor subtypes suggest that the IL-12R in the eosinophils can
be functional.
IL-12 primarily regulates the balance between Th1 and Th2 cells
(16, 25, 26, 27). IL-12 induces Th1 cell differentiation and
proliferation, and contributes to optimal production of IFN-
(13, 14). Besides this activity of IL-12 in inducing the
Th1-type responses, IL-12 was also reported to inhibit Th2-type
responses. IL-12 inhibited allergen-induced hyperresponsiveness and
eosinophil accumulation in murine (18, 19, 21) models of
asthma, and Ag challenged human airways (B. J. Connor, unpublished
observation). In a recent study, Naseer et al. (22)
demonstrated that IL-12 mRNA expression was decreased within the
airways of asthmatic subjects compared with those of nonasthmatic
control subjects. These findings demonstrated the relative deficiency
of IL-12 in asthmatic airways, which could be due to the overexpression
of IL-4 (27, 34). The inhibitory effects of IL-12 on
eosinophil numbers and airway hyperresponsiveness can also be
attributed to IL-12-mediated inhibition of Th2-promoting cytokines such
as IL-4 (16, 18, 20). These previous reports do not,
however, preclude other functions for this cytokine. Taken together
with our current data, the lower levels of IL-12 in asthmatics suggest
that there may be less eosinophil apoptosis and thus less turnover of
the receptor compared with nonasthmatic subjects. This may explain the
discrepancy between the IL-12R mRNA levels detected in normal control
subjects as compared with asthmatics.
To date, studies demonstrate the capacity of eosinophils to express predominantly Th2-type cytokine receptors both constitutively and after appropriate stimulation. IL-5 is the primary growth factor in eosinophils (35), and they also respond to IL-3, GM-CSF (36), and IL-9 (37). In the present study, IL-12R, known as a modulator of Th1 responses, was detected constitutively on the majority of eosinophils from both asthmatic and normal control subjects. We were unable to detect any difference in protein expression or in response to IL-12 in eosinophils derived from asthmatics as compared with nonasthmatic control patients.
Using flow cytometry, baseline IL-12R surface expression was detected in four of eight eosinophil preparations obtained. In all other subjects, cytoplasmic IL-12R expression determined by immunocytochemistry (data not shown). One explanation for the discrepancy between the results using these two methods is the sensitivity of detection of low surface expression in some individuals (38). This is supported by the fact that when eosinophils with low surface receptor expression were stimulated with PMA (data not shown), the expression of IL-12R was significantly induced and thus detectable by FACS analysis. This is further supported by acceleration of programmed cell death in response to IL-12 administration seen in all subjects.
The mechanism by which PMA up-regulated the surface expression of
IL-12R is unknown. A possible mechanism is throughinduction of
cytokines, including IL-12 or IFN-. However, none of these cytokines
or IL-5 and IL-18 augmented surface expression of IL-12R. It is unclear
whether the induction of surface IL-12R by PMA changes the functional
response to IL-12. These studies are ongoing in our laboratory.
In culture, in the absence of growth-promoting cytokines, eosinophils
gradually undergo programmed cell death. We propose that the
well-documented decrease in eosinophil numbers in response to IL-12
is due to the acceleration of this process. Morphological analysis of
apoptotic nuclei and DNA fragmentation determined by PI staining were
consistent with this hypothesis (
Figs. 5–7). This effect is dose
dependent, with effects between 1 and 100 ng/ml (Fig. 6). Although
previous studies have demonstrated a role for IL-12 in defined
Fas-mediated apoptosis in T cells (39, 40), our data
provide the first report demonstrating direct effects of IL-12 on
eosinophil apoptosis.
The pathway by which IL-12 induces in vitro eosinophil apoptosis is not
clear. Our preliminary results suggest that neither IL-12 have any
effects on FAS expression on eosinophils (data not shown). Similarly,
IL-12 does not increase TNF-
receptor expression on eosinophils,
determined by FACS analysis (E. Nutku and Q. Hamid, unpublished
observation), nor does it decrease the expression of IL-5 mRNA (R.
Schleimer, unpublished observation). IL-5 serves as a survival
factor for eosinophils (8, 9, 10). When mature peripheral
blood eosinophils are cultured in the absence of IL-5, they undergo
apoptosis. In the present study, IL-5 (2.5 ng/ml) rescued eosinophils
from the apoptotic effect of IL-12 (Fig. 8). The mechanism of IL-5 and
IL-12 interaction is not clear. IL-5 may act by decreasing inhibition
of IL-12R expression, rendering the eosinophils unresponsive to IL-12.
However, IL-5 had no regulatory effects on eosinophil-IL-12R
expression.
Our time course experiments (Table I) provided new perspectives to
explain IL-5 and IL-12 interaction in association with eosinophil
apoptosis. The presence of either of the cytokines in initial
eosinophil cultures reversed the effect of the other on eosinophil
apoptosis. However, addition of either IL-5 or IL-12 at later time
points of the cell culture did not affect the outcome. These data may
imply that IL-5 and IL-12 use different pathways to inhibit or to
accelerate the eosinophil apoptosis, respectively. Stimulation of
eosinophils for at least 3 h appeared to commit the cells to
programmed cell death. This is consistent with the up-regulation of new
proteins including caspases and Bcl family proteins involved in
apoptotic processes (41, 42). Whether these proteins are
associated with IL-12 effects on eosinophil apoptosis is still to be
determined.
IL-12 has been shown to have important effects on other cells
including T cells and NK cells. This includes induction of IFN- and
other proinflammatory cytokines (14, 43). In addition, it
has been demonstrated to decrease the synthesis of IgE (17, 20). Although eosinophils are known to produce cytokines
including IL-12, (44), we do not believe that exogenous
IL-12 augments production of cytokines such as IL-12 or IFN-, as the
signal from IL-12 appears to rapidly commit eosinophils to programmed
cell death. In addition we cannot exclude that IL-12 may have some
effect on eosinophil progenitors, either to enhance or retard cell
differentiation. This hypothesis is currently being tested in our
laboratory.
Our findings suggest a mechanism by which IL-12 exerts an inhibitory effect on eosinophils in allergic and parasitic inflammation (16, 17, 18). Our experiments also demonstrate that the critical balance between dendritic cell derived cytokines such as IL-12 and Th2-derived cytokines including IL-5 in the microenvironment of an allergic reaction play a crucial role in determining the survival and effect of eosinophils in inflamed tissues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Qutayba Hamid, Meakins Christie Laboratories, McGill University, 3626 Saint Urbain Street, Montreal, Quebec, Canada H2X 2P2. E-mail address: hamid{at}meakins.lan.mcgill.ca
3 Abbreviation used in this paper: PI, propidium iodide.
Received for publication November 8, 2000. Accepted for publication May 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and interleukin-10. J. Exp. Med. 183:2559. type cytokine receptor subunits. Proc. Natl. Acad. Sci. USA 93:14002.2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185:817. subunit gene. J. Biol. Chem. 270:1462.This article has been cited by other articles:
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, p < 0.005, IL-12 vs IL-5) or medium
alone (*, p < 0.05, medium vs IL-12). Data are
mean percent of apoptotic cells ± SEM; n =
5.
