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Departments of
*
Internal Medicine and
Pathology, Virginia Commonwealth University, Richmond, VA 23298; and
Department of Metabolic Diseases, Hoffman-LaRoche Inc., Nutley, NJ 07110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, and IL-4R on their surface. Mast cells
examined after purification by immunomagnetic sorting became apoptotic
within hours after exposure to rhIL-4, a phenomenon blocked by
anti-IL-4 Ab. Because rhIL-4-dependent apoptosis but not the loss
of mitochondrial membrane potential was prevented by the pan-caspase
inhibitor benzyloxycarbonyl-Val-Ala-Asp-(Z-VAD)-fluoromethylketone,
mitochondrial perturbation most likely preceded caspase activation.
Consistent with this conclusion was the observation that both apoptosis
and loss of mitochondrial membrane potential (
m) were
inhibited by cyclosporin A in combination with aristolochic acid.
rhIL-6 protected cord blood mast cells from rhIL-4-induced apoptosis.
Thus, IL-4 can cause both maturation and apoptosis of human mast cells,
the latter effect being abrogated by IL-6. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI
(8), those derived from cord blood, peripheralblood, and
bone marrow, in most but not all cases, express surface FcRI when
rhSCF is the sole exogenous growth factor. rhIL-4 influences human mast
cell development in various ways. For example, when added to the human
mast cell leukemia line, HMC-1, rhIL-4 down-regulates surface Kit
expression (9). rhIL-4 added with rhSCF to fetal liver
cells at the beginning of culture results in diminished surface levels
of Kit on the mast cells that develop, a modest decline in the numbers
of mast cells obtained (10), and induction of expression
of functional surface FcRI that is associated with a 10-fold
increase in cellular levels of FcRI
mRNA (11). Mast
cells derived from cord blood progenitors in the presence rhIL-4
together with rhSCF and rhIL-6 showed increased chymase expression
(12), whereas those derived from fetal liver progenitors
in the presence of rhIL-4 and rhSCF showed a decrease in the number of
the chymase-, MCT type of
mast cell, with no change in number of the
chymase+, MCTC type of mast
cell (11). For mast cells derived from human cord blood in
the presence of rhSCF and rhIL-6, rhIL-4 induces homotypic aggregation
dependent upon LFA-1 and ICAM-1 (13), and detectable
levels of surface FcRI (14). rhIL-13, like rhIL-4 but
weaker, down-regulates Kit expression and up-regulates LFA-1 and ICAM-1
on HMC-1 cells, but has a negligible effect on the development of mast
cells from cord blood progenitors exposed to rhSCF
(15). The present study examines the effects of rhIL-4 on the development of rhSCF-dependent human mast cells from cord blood mononuclear cells (CBMC) and shows that rhIL-4 can induce apoptosis via a mitochondrial-dependent pathway in the mast cells that develop in the absence of rhIL-6, whereas cord blood-derived mast cells are protected from rhIL-4-mediated apoptosis if developed in the presence of rhIL-6.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Umbilical cord blood was obtained at the time of delivery and collected in heparin-treated tubes. The experimental protocol was approved by the Human Studies Committee at Virginia Commonwealth University (Richmond, VA). Cord blood was diluted 1:1 in PBS, layered over Histopaque (density = 1.077 g/ml; Sigma, St. Louis, MO) and centrifuged at 1000 x g for 20 min at room temperature to remove erythrocytes. CBMC were collected from the interface, washed twice in PBS, and submitted to another density-dependent centrifugation step as described above. Cells at the interface were collected, washed three times in PBS, and suspended in RPMI 1640 supplemented with 10% heat-inactivated controlled process serum replacement medium-1 (Sigma), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 10 mM HEPES, 50 µM 2-ME, 200 U/ml penicillin, and 100 µg/ml streptomycin. Cells were dispensed into 24-well plastic tissue culture plates (Costar, Cambridge, MA) at 106 cells/2 ml/well in the presence of rhSCF (100 ng/ml; a gift from Amgen, Thousand Oaks, CA) alone or together with different concentrations of rhIL-4 (a gift from Amgen) and with rhIL-6 (50 ng/ml; Genzyme, Cambridge, MA). Cells were cultured at 37°C in a 6% CO2 incubator. Half of the culture medium was replaced once a week. Cells were harvested at different time points and subjected to cytocentrifugation and to flow cytometry.
In some cases, fetal liver-derived mast cells and cord blood-derived cells were cocultured. Fetal liver-derived mast cells were prepared as described previously (1); complete medium containing rhSCF (50 ng/ml) was added every 3–4 days over a 4- to 6-wk time span. Cord blood cells were cultured as above with rhSCF (100 ng/ml) and rhIL-4 (20 ng/ml), also for 4–6 wk. Cells were then washed twice with PBS, counted, and cultured with rhSCF (100 ng/ml) in the presence and absence of rhIL-4 (20 ng/ml) for 6 days at 106 total cells/ml; the cells consisting of fetal liver-derived cells alone, cord blood-derived cells alone, or a 1:1 mixture of fetal liver- and cord blood-derived cells. Total cell numbers and viabilities and the number of Kit+ cells were determined in each case.
Surgical lung tissue samples (10–60 g from patients with lung cancer
or emphysema) were obtained from the Department of Pathology at
Virginia Commonwealth University or through the Cooperative Human
Tissue Network (Columbus, OH) as approved by the Human Studies
Institutional Review Board at Virginia Commonwealth University. Each
sample was minced extensively and then digested with a combination of
type IA collagenase and type I-S hyaluronidase (Sigma; 1.5 and 0.75
mg/ml, respectively) in MEM + 2% FCS containing penicillin (200 U/ml),
streptomycin (100 µg/ml), and amphotericin B (500 ng/ml). Four
milliliters of enzyme solution was applied per gram of tissue and
incubated for 30 min at 37°C with gentle shaking. The dispersed cells
(D1) were separated from residual tissue by filtration through a
70-µm mesh nylon sieve. The remaining tissue was minced and digested
again as above. Cells from the second digestion (D2) were collected and
filtered as above. Filtered cells were washed three times in MEM + 2%
FCS (500 x g for 8 min at room temperature). Depending
on their respective mast cell yields, D1 and D2 were pooled. Mast cells
were detected on cytospins stained with acidic toluidine blue (0.5% in
0.5 N HCl). Total cell viability was determined by trypan blue
exclusion. Erythrocytes were eliminated from the cell preparation by
centrifugation over a continuous 65% Percoll gradient (Pharmacia,
Piscataway, NJ). After each purification step (D1, D2, and Percoll
gradient), the viability and the mast cell content of each fraction was
monitored as described above. Cells (15–20% mast cells) were then
cultured overnight in an incubator at 37°C with 6%
CO2, in the same medium used to culture cord
blood cells at a final concentration of 2 x
106 total cells/ml. Cells were washed and placed
into culture for 2 days in the same medium containing rhSCF (100 ng/ml)
in the absence or presence of rhIL-4 (20 ng/ml). Cells were analyzed by
flow cytometry to determine the level of expression of Kit, FcRI,
and IL-4R.
Flow cytometry
Human cord blood-derived cells were analyzed for expression of
surface Kit using the mAb YB5.B8 (a gift from Dr. Leonie Ashman,
Institute of Medical and Veterinary Science, Adelaide, Australia),
FcRI using mouse IgG1 mAbs 29C6 and 15A5 against the FcRI
subunit (16), or a mouse IgG1 mAb against the -chain of
the IL-4R (catalog no. 80-3285-01; Genzyme). Cultured CBMC
(105) were washed once in PBS and incubated in
DMEM containing 10% human AB serum for 30 min at 4°C. Cells were
washed once in PBS containing 1% BSA and 0.1% sodium azide and were
incubated at 4°C for 30 min in 100 µl of PBS containing 1% BSA,
0.01% thimerosal, and either YB5.B8 (ascites diluted 1/1000), purified
29C6 (2 µg/ml), anti-IL-4R (10 µg/ml), or an IgG1
isotype-matched, negative control mAb, MOPC-31C (2 or 10 µg/ml).
Cells were washed twice, and incubated for 30 min at 4°C in 100 µl
of diluent containing 100 µg/ml of rabbit IgG. After a wash, cells
were incubated with FITC-labeled rabbit F(ab')2
anti-mouse IgG (1/40 dilution; Dako, Copenhagen, Denmark) at 4°C
for 30 min, washed twice, and analyzed with a FACScan (Becton
Dickinson, San Jose, CA). Cell lines (HMC-1 and KU-812) were stained in
parallel as positive controls (HMC-1 for Kit and IL-4R and KU812 for
FcRI and IL-4R). HMC-1 was a gift from Drs. G. Gleich and J.
Butterfield (Mayo Clinic, Rochester, MN) (17). The human
basophil leukemia cell line, KU812, was obtained from Dr. G. Nilsson
(University of Uppsala, Uppsala, Sweden) (18). Just before
the flow cytometry, propidium iodide was added to each cell suspension
so that dead cells could be excluded. The net percentage of positive
cells was determined by subtracting the percentage of cells stained
with the negative control (
10% for IL-4R and FcRI and <1% for
high Kit) from the percentage of positive cells stained with each
relevant mAb. Although 
2% of the starting population of CBMC were
Kit+ at a low mean fluorescence intensity, only
cells with a high mean fluorescence intensity (>10-fold above
background mean fluorescence intensity) were scored as
Kit+ in the current study. The analysis was
performed using the CELLQUEST software (Becton Dickinson).
In some experiments, cells were double-labeled for Kit and either
FcRI or IL-4R. Cells were first labeled with either 29C6
(anti-FcRI mAb) or anti-IL-4R mAb, displayed with
FITC-rabbit F(ab')2 anti-mouse IgG, blocked
with mouse IgG, and then labeled and displayed with PE-conjugated
YB5.B8 mAb (PharMingen, San Diego, CA). PE-conjugated mouse IgG1 mAb
was used as an isotype-matched negative control (Becton Dickinson).
Immunomagnetic purification of Kit+ cells
CBMCs were cultured for 7 wk in the presence of rhSCF (100 ng/ml). The isolation of Kit+ cells was adapted from a method previously described (19). Briefly, 106 cells were pelleted, resuspended in 10 ml of DMEM containing 10% human AB serum and 100 µg/ml mouse IgG for 30 min at 4°C, washed twice in ice-cold PBS, washed once in HBSS containing 2% FCS, resuspended in 1 ml of a 1:100 dilution of YB5.B8 ascites fluid in HBSS containing 2% FCS, incubated for 30 min at 4°C with gentle shaking, washed twice and resuspended in HBSS containing 2% FCS, and then incubated with Dynabeads coated with a sheep anti-mouse IgG (Dynal, Oslo, Norway) in a volume of 1 ml for 1 h with gentle shaking (Dynabead:Kit+ cell ratio = 6:1). The suspension was then diluted with 7 ml of HBSS containing 2% FCS and placed within the field of an MPC-1 magnet (Dynal) for 5 min. Free Dynabeads and Kit+ cells attached to the Dynabeads adhere to the side of the tube. Unattached Kit- cells were washed away. Attached cells were washed with 20 ml of HBSS containing 2% FCS and again placed within the magnetic field. Unattached cells were pooled with the previous collected portion of Kit- cells. Kit+ cells were put in culture with complete RPMI for 2 days to allow the beads to detach. Kit- cells were cultured in parallel. Among the cells isolated in the Kit+ fraction, 95–99% were stained by immunofluorescence with the G3 mAb against tryptase (see below), a marker for mast cells.
Immunocytochemistry
Cytocentrifuge preparations of cells were fixed in methanol containing 0.6% H2O2 for 30 min at room temperature, rinsed with H2O, and stored at 4°C until used. Slides were labeled with biotin-conjugated B7 (or biotin-MOPC, as an isotype-matched negative control), a mouse IgG1 antichymase mAb, and alkaline phosphatase-conjugated G3 (or alkaline phosphatase-MOPC-31C), as previously described, to identify MCTC cells (chymase+ and tryptase+) that stain reddish brown with 3-amino-9-ethylcarbazole, and MCT cells (tryptase+ and chymase-) that stain blue with Fast Blue RR (Sigma) (20). Because no chymase+ MCTC cells were detected, only G3 was used in double-staining experiments aimed at examining apoptosis in mast cells or of IL-4R expression in mast cells. In the latter case, slides were first incubated with PBS containing 10% normal rabbit serum, 1% BSA, and 0.01% thimerosal for 1 h at room temperature. Slides were then washed in H2O and incubated overnight at 4°C with either goat polyclonal IgG anti-human IL-4R (10 µg/ml; R&D Systems, Minneapolis, MN) or nonimmune goat IgG as a negative control (10 µg/ml). Slides were washed three times for 5 min each time in 0.01 M Tris buffer, pH 7.4, containing saline and 0.05% Tween 20 (TTBS), rinsed with H2O, and incubated for 1 h at room temperature with a biotin-conjugated rabbit IgG anti-goat IgG (7.6 µg/ml; Sigma). Slides again were washed three times for 5 min in TTBS, rinsed with H2O, and incubated with streptavidin-peroxidase conjugate (20 µg/ml) for 1 h at room temperature. Slides were washed as described above, rinsed with H2O, and incubated with 3-amino-9-ethylcarbazole/0.01% H2O2 for 7 min at room temperature. The reaction was stopped by rinsing the slides with H2O. Slides were then processed for the immunofluorescence staining of tryptase using tetramethylrhodamine isothiocyanate (TRITC)-G3 (or a TRITC-MOPC-31C isotype-matched negative control), kindly provided by Dr. Angela Hogan (Virginia Commonwealth University), at a concentration of 10 µg/ml. After an overnight incubation at 4°C, slides were washed in TTBS and analyzed using a BX50 fluorescence photo microscope with a PM-30 exposure control attachment (Olympus Optical, Tokyo, Japan).
In situ detection of apoptotic mast cells
The identification of apoptosis at a single cell level on slides was performed using the TACS in situ apoptosis detection kit according to the manufacturer (Genzyme). The principle of the technique is to detect DNA fragments generated during apoptosis. Biotinylated nucleotides are incorporated into the ends of DNA fragments using TdT. Labeled ends are detected using streptavidin-HRP and the peroxidase substrate, TACS Blue Label (Genzyme), which stains apoptotic cells blue. Pelleted cells were gently suspended in 10% neutral-buffered formalin at a concentration of 106 cells/ml and incubated for 10 min at room temperature. Fixed cells were centrifuged and resuspended in 80% ethanol. At room temperature, 50 µl of this cell suspension was dropped onto a glass slide, air-dried for 5–10 min, incubated with 70% ethanol for 5 min, air-dried for 1 h, and treated with 1 µg of proteinase K for 5 min after rehydration. To quench endogenous peroxidase activity, slides were placed in a Coplin jar containing a solution of 2% H2O2 for 5 min at room temperature. Slides were then transferred to a solution of 1x labeling buffer containing TdT dNTPs (1x final concentration) and adjusted to 1 mM Co2+. Labeling was initiated by addition of 15 U of TdT per sample. The labeling reaction was allowed to proceed for 15 min at 37°C and was then stopped by transferring the slides into a solution of TdT stop buffer. Slides were then rinsed for 1 min at room temperature in water, incubated with 10 ng of streptavidin-HRP for 10 min at room temperature, washed twice with water, and incubated with 50 µl of TACS Blue Label. To decide when to stop the staining reactions, the staining of control slides provided by the manufacturer and developed in parallel were monitored microscopically. Typically, after about 7 min at room temperature the slides were washed four times in water, each wash lasting 5–10 s. Slides were then processed for the immunofluorescence staining of tryptase using TRITC-G3 and analyzed as described above. For each experiment, four different slides of each culture condition were processed and a total of 100–200 cells were analyzed per slide. Slides were read by one observer in a blinded fashion. DNase-free deionized water was used throughout the procedure.
Where indicated, annexin V (Genzyme) also was used to detect apoptotic cells. Harvested cells to analyze were pelleted by centrifugation and washed in cold PBS. Cell pellets were resuspended in a 1:100 dilution of annexin V-biotin, and developed according to the manufacturer’s instructions. As a negative control, cells from the same sample were incubated with MOPC-biotin.
Measurement of changes in mitochondrial membrane potential
(m)
Purified Kit+ cord blood-derived mast
cells (105/250 µl of complete RPMI 1640 medium)
were loaded with 40 nM of the lipophilic, cationic, and fluorescent
dye, 3,3'-dihexyloxacarbocyanine iodide
(DiOC6(3); Molecular Probes, Eugene, OR)
(21, 22) for 20–25 min at room temperature before being
analyzed. DiOC6(3) accumulates in intact
mitochondria, but levels are reduced in cells undergoing the
permeability transition accompanied by loss of
m. Mitochondrial retention of
DiOC6(3) was determined up to 6 h after
treatment in the presence of rhSCF (100 ng/ml) alone or with rhIL-4 (20
ng/ml), using a FACScan (Becton Dickinson) and CYCLOPS
software. In addition, the following treatments (all reagents were
purchased from Biomol Research, Plymouth Meeting, PA, and were
dissolved in DMSO) were assayed on the same purified cord blood-derived
mast cellsbefore the treatment with rhIL-4. The pan-caspase inhibitor
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.FMK) was added
to the cells at 25 µM, 1 h before rhIL-4 (23).
Cyclosporin A (CyA) was added to the cells at 2.5 µM and FK506 at 5
µM, each one together with 50 µM aristolochic acid (ArA) and 30 min
before treatment with rhIL-4 (24). This dose of CyA in
combination with ArA was the highest found to be noncytotoxic to the
cells. A portion of the cells from each experimental group was assessed
for apoptotic DNA fragmentation, as described above (n
= 4–6).
Statistical analysis
Statistical analyses were performed using SigmaStat (Jandel, San Rafael, CA). Data sets were first tested for normality, and then statistical comparisons were performed by ANOVA for data sets having normal distributions and by a nonparametric Mann-Whitney rank test for data sets not having a normal distribution. Statistically significant values were considered to be p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of cell surface FcRI and Kit were analyzed weekly by
flow cytometry on CBMC cultured either in the presence of rhSCF alone
(100 ng/ml) or in combination with rhIL-4 (20 ng/ml). These conditions
were chosen to mirror those used to develop mast cells from fetal liver
cells in our lab (11) and as reported by others for
development of mast cells from CBMC (2) and peripheral
blood and bone marrow cells (5). This dose of rhIL-4 was
previously shown to increase expression of FcRI on rhSCF-dependent
mast cell development from fetal liver cells while also causing a
modest decline in mast cell numbers (11). IL-6 has been
used to enhance rhSCF-dependent development of cord blood-derived mast
cells in other labs, and additional treatment of such cells with rhIL-4
been shown to promote features such as chymase and FcRI expression
(7, 12, 13, 14, 25, 26). The effect of rhIL-6 in our system on
the IL-4 response will be considered below.
Kit+ and FcRI+ cells
appeared 2 wk after cultures were initiated with rhSCF alone or
together with rhIL-4. As shown in Fig. 1
A, inclusion of rhIL-4 caused
a marked (7- to 8-fold) increase in cell number at 2, 4, and 8 wk of
culture relative to conditions using rhSCF alone. Although the
percentages of Kit+ and
FcRI+ cells were significantly greater in the
absence of rhIL-4 than in its presence at 2, 4, and 8 wk of culture
(Fig. 1B), absolute numbers of Kit+
and FcRI+ cells were no different in the
presence and absence of rhIL-4 at 2 wk. However, by 4 wk and also at 8
wk of culture, the numbers of Kit+ cells and
FcRI+ cells were significantly lower in the
presence than in the absence of rhIL-4. In fact, almost no
Kit+ or FcRI+ cells
could be detected at 8 wk. These results suggested that rhIL-4 acted to
expand nonmast cell populations and to decrease numbers of mast cells
after they had formed but did not prevent mast cell formation. Also,
the effects of rhIL-4 on Kit+ and
FcRI+ cells occurred in parallel. If addition
of rhIL-4 was delayed until day 7 or day 14, and the cells were
examined at 4 or 8 wk, almost no FcRI+ and
Kit+ cells were detected, suggesting that rhIL-4
was affecting the survival of newly formed mast cells (data not
shown).
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RI+ cells developing from CBMC is shown in
Fig. 2. FcRI+
and Kit+ cells after 8 wk of culture with rhSCF
and different doses of rhIL-4 decreased in parallel with increasing
doses of rhIL-4. Essentially no receptor-positive cells were detected
at 2 and 20 ng/ml of rhIL-4. Because the total numbers of cells
remaining after 8 wk of culture were higher with increasing
concentrations of rhIL-4, the EC50 concentrations
of rhIL-4 for mast cell number and mast cell percentage differed. The
EC50 for mast cell percentage was about 0.01
ng/ml, whereas the EC50 for mast cell number was
about 0.1 ng/ml.
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RI+
mast cells
Because rhIL-4 appeared to act primarily on mast cells rather than
mast cell progenitors, rhIL-4 (20 ng/ml) was added along with rhSCF to
mast cells that had formed after 7 wk of culture of CBMC with rhSCF.
Cells were examined daily for FcRI by flow cytometry over a 4-day
span and the results of three independent experiments are shown in Fig. 3. In the presence of rhSCF alone at 100
ng/ml, the percentage of cells positive for FcRI was approximately
the same on days 4 and 0 (data not shown). In the presence of rhIL-4
there was a progressive decline in the numbers and percentages of cells
expressing FcRI. Despite the decline in mast cells, total cell
numbers increased from a mean of 3.5 x 105
to a mean of 6.2 x 105 over the 4-day
experiments.
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To determine whether rhIL-4 might directly exert its effects on
cord blood-derived mast cells, the expression of IL-4R was analyzed by
flow cytometry and immunocytochemistry. CBMC were cultured for 7 wk in
rhSCF alone (100 ng/ml) and then divided into two portions. One portion
was cultured again in rhSCF alone, the other in the presence of both
rhSCF and rhIL-4 (0.002, 0.2, and 20 ng/ml). One wk later, cells from
each experimental condition were analyzed for cell surface Kit and
IL-4R by flow cytometry and cellular tryptase and IL-4R by
immunocytochemistry (Fig. 4A).
Interestingly, the percentages of cells cultured in rhSCF alone that
expressed IL-4R were not significantly different from the percentages
expressing Kit and tryptase, each being detected on about 50% of the
cells. Dual labeling of cytospins by immunocytochemistry indicated that
>95% of the rhIL-4+ cells and >95% of the
tryptase+ cells were double-positive when cells
were cultured in rhSCF alone.
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To determine whether surface Kit, FcRI, and IL-4R were expressed on
the same cell population, double-labeling experiments were performed,
as shown in Fig. 4
B. In this figure representative dot blots
are shown of CBMC that had been cultured for 7 wk with rhSCF (55%
tryptase+ cells) and then exposed to rhSCF alone
(Fig. 4B, upper) or to rhSCF and rhIL-4 (Fig. 4B, lower) for only 2 days before analysis.
Without treatment with rhIL-4, Kit+ cells
accounted for about 41% of the total, and nearly all were also
FcRI+ (Fig. 4B, upper
left) and IL-4R+ (Fig. 4B,
upper right). FcRI+ or
IL-4R+ cells with low levels or no detectable Kit
accounted for about 38% of the cells. Although these cells could
represent nonmast cell lineages, it is more likely that most of these
cells represent mast cells that had internalized Kit-rhSCF complexes,
as reported previously (27). As shown above, by 4–7 days
after addition of rhSCF, nearly equal percentages of cells are positive
for surface Kit, IL-4R, and FcRI. Addition of rhIL-4 together with
rhSCF for 2 days resulted in a dramatic decrease in the percentages of
surface Kit+/IL-4R+ and
Kit+/FcRI+ cells (Fig. 4B, lower) to <1. The percentages of
Kit-/FcRI+ and
Kit-/IL-4R+ cells
increased to about 62%, but the total percentages of
FcRI+ and IL-4R+ cells
decreased from about 78 to 63%. Again, it is likely that rhSCF-induced
internalization of Kit accounted for the disproportionate decrease in
Kit+ cells. It is also possible that rhIL-4 may
have delayed recovery of surface Kit.
Mast cells incubated in the presence of rhIL-4 for 2 days undergo apoptosis
To evaluate whether apoptosis could account for the decreased
numbers of mast cells observed in the presence of rhIL-4, cells were
double labeled for apoptotic fragmentation of DNA in nuclei and for
tryptase in mast cell secretory granules. Fig. 5A shows a fluorescence
photomicrograph of tryptase+ mast cells, whereas
Fig. 5B shows a light photomicrograph of the same field of
cells stained for excessive DNA fragmentation; these cells had been
cultured with rhSCF alone for 5–7 wk. As summarized in Table I, in two independent experiments none of
the tryptase+ cells in preparations of cord
blood-derived mast cells exposed to rhSCF alone appeared to be
apoptotic, whereas 40–50% of the tryptase-
cells showed DNA fragmentation by the TACS Blue Label procedure,
consistent with apoptosis. During this 2-day culture there were modest
declines (20–30%) in total cell numbers with these unsorted
preparations of mast cells. Fig. 5, C and D, are
higher magnification photomicrographs of a single cell from a culture
treated with rhSCF for 7 wk and rhSCF and rhIL-4 (20 ng/ml) for 2 days.
In this case, the cell is tryptase+ (Fig. 5C) and appears to be undergoing apoptosis (Fig. 5D). The overall percentage of apoptotic cells 2 days after
addition of rhIL-4 remained about the same as for those cells exposed
only to rhSCF. However, the percentages of mast cells undergoing
programmed cell death increased dramatically when rhIL-4 was added,
increasing from 0 to 94% in one experiment and from 0 to 70% in a
second experiment. Increased apoptosis of mast cells was associated
with declines in the percentages and calculated numbers of
tryptase+ mast cells, indicating a selective loss
of mast cells during the 2-day incubation with rhIL-4.
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95% mast cells as
judged by their tryptase+ staining. As reported
in Table I, two independent experiments showed that the addition of
rhIL-4 to purified cord blood-derived mast cells induced apoptosis
among the mast cells. Within 2 days of culture, rhIL-4 caused >90% of
the mast cells to become apoptotic, coupled with 3- to 4-fold decreases
in the percentages and 30- to 38-fold decreases in the total numbers of
tryptase+ cells. rhIL-4-mediated apoptosis of cord blood-derived mast cells is blocked by anti-IL-4 Ab
To validate that rhIL-4 was responsible for the apoptosis observed
above, mast cells derived from cord blood progenitors in the presence
of rhSCF were treated for 3 h under the conditions shown in Fig. 6. This period had been determined to
result in apoptosis of about 30% of the mast cells without
substantially reducing the number of mast cells. As shown, the
apoptotic effect of rhIL-4 was significantly reduced by neutralizing
anti-IL-4 Ab but not by control Ab.
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m is a primary event during
rhIL-4-induced apoptosis of cord blood-derived mast cells
Induction of apoptosis in many systems has been related to
initiation of mitochondrial damage, including loss of the
m and/or release of cytochrome c
into the cytoplasm (24). To determine whether an
alteration in m or activation of upstream
caspase(s) was the primary event during rhIL-4-induced apoptosis of
purified cord blood-derived mast cells, a general inhibitor of caspase
activity and an antagonist of mitochondrial permeability transition
were utilized. rhIL-4 applied at 20 ng/ml for 6 h resulted in a
decreased m in 32% of the cells and
apoptosis (by DNA fragmentation) in 18% compared with medium controls
(Table II). No alteration in
m or the extent of apoptosis was detected
between rhIL-4-treated and untreated cells 1 h after addition of
this cytokine, whereas by 3 h there were significant alterations
in each parameter which increased further by 6 h (data not shown).
Fig. 7 shows a representative flow
cytogram of an experiment in which 40% of the cells showed a decrease
in m 6 h after exposure to rhIL-4. To
inhibit caspase activation, Z-VAD.FMK was added. Z-VAD.FMK abrogated
apoptosis assessed by DNA fragmentation, as anticipated, but did not
prevent the loss of m (Table II and Fig. 7), indicating that the loss of m was not
dependent on activation of the upstream activator caspases, including
caspase-8 (28). Apoptosis as well as the loss of
m induced by rhIL-4 were prevented by
pretreatment with CyA in combination with ArA, agents known to prevent
the mitochondrial permeability transition induced directly by Bax
(24). However, this effect was unrelated to the ability of
CyA to inhibit calcineurin, because FK506 (5 µM), another calcineurin
inhibitor, in combination with ArA, did not prevent either of these
events (Table II). Together, these findings suggest that exposure of
cord blood-derived mast cells to rhIL-4 results in loss of
m, which in turn leads to activation of the
apoptotic protease cascade.
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Fetal liver-derived mast cells (4–5 wk of culture with rhSCF
alone) of 67 ± 20% purity (n = 5) were examined
by flow cytometry for the presence of IL-4R. Only 1.9 ± 1.5% of
the total number of cells were positive. As shown in Fig. 8, exposure of such mast cells to rhSCF
(100 ng/ml) and IL-4 (20 ng/ml) for 6 days resulted in no significant
change in the number of Kit+ mast cells.
Examination of these cells for apoptosis by our double-labeling
technique also revealed essentially no apoptotic mast cells in either
case. To determine whether cord blood cells obtained after 4–6 wk of
culture with SCF and IL-4 would alter the response of fetal
liver-derived mast cells to IL-4, coculture experiments were performed.
Such cord blood-derived cells were 80% CD3+,
with about one-third of those being
CD4+CD8+ and the remainder
being equally divided between
CD4+CD8- and
CD4-CD8+ cells. Less than
4% of the cells were mast cells. Equal numbers of fetal liver-derived
cells (67% mast cells on average) and cord blood-derived cells (1%
mast cells on average) were cocultured for 6 days in medium containing
rhSCF (100 ng/ml) with or without rhIL-4 (20 ng/ml). As shown in Fig. 8, addition of rhIL-4 to rhSCF during this 6-day coculture period
failed to decrease the number of mast cells as evidenced by the number
Kit+ cells being unaltered. This result is
dramatically different from that obtained with cord blood-derived mast
cells (Table I). Thus, fetal liver-derived mast cells do not undergo
IL-4-mediated apoptosis and do not develop this response under our
experimental coculture conditions.
|
RI, and IL-4R. Total cell numbers recovered in
the absence (5.8 x 105 ± 2.3 x
105) and in the presence (5.9 x
105 ± 2.3 x 105) of
rhIL-4 were no different (n = 6) using paired
Student’s t-test analyses corrected for the number of
comparisons; neither were the respective percentages of
FcRI+ (32 ± 3 and 28 ± 6) and
Kit+ (33 ± 4 and 29 ± 5) cells, or
the respective numbers of tryptase+ mast cells
(1.5 x 105 ± 0.5 x
105 and 1.1 x 105 ±
0.6 x 105). Like cord blood-derived mast
cells, essentially all human lung-derived mast cells appeared to be
IL-4R+ by double-labeling with PE-YB5.B8. Mast cells developed with rhSCF and rhIL-6 are protected from rhIL-4-mediated apoptosis
Because the observation of apoptosis to rhIL-4 differs from prior
reports in which rhIL-6 was used along with rhSCF to develop mast cells
from CBMC, experiments were performed to examine the effect of rhIL-6
on rhIL-4-mediated apoptosis. The Kit+ cord
blood-derived mast cells obtained in the presence of rhSCF with or
without rhIL-6 were purified using immunomagnetic beads, detached
during 1 wk of culture under the same conditions, and exposed to rhIL-4
for 3 h. Annexin V-biotin, which detects apoptosis at an earlier
stage than the TACS procedure, as well as the TACS procedure, were used
to examine apoptosis. As shown in Fig. 9A, rhIL-4 induced apoptosis
in about 23% of the rhIL-6 naive cells, whereas the apoptosis
percentage in rhIL-6-treated cells was about 5%, which was not
significantly different from the rhIL-6 naive or rhIL-6-treated cells
that were not exposed to rhIL-4. Additional experiments showed that
rhIL-6 could be added to cultures of cord blood-derived mast cells and
rhSCF after 5–7 wk, and within 1 day provided protection from
rhIL-4-mediated apoptosis (data not shown). Thus, rhIL-6 protects
developing cord blood-derived mast cells from rhIL-4-mediated
apoptosis.
|
B. However, no further decrease in IL-4R expression was
noted after 24 h. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of rhIL-4 appeared to be direct, because double labeling
indicated that all Kit+ mast cells were
FcRI+ and IL-4R+ and
because highly purified cord blood-derived Kit+
mast cells also underwent apoptosis when exposed to rhIL-4. However,
indirect effects of rhIL-4 in the mixed cell cultures cannot be
completely excluded. The apoptotic effect of rhIL-4 is unlikely to be
due to diminished levels of surface Kit. Kit levels on
rhSCF-dependent fetal liver-derived human mast cells, bone
marrow-derived mast cells, and HMC-1 cells showed only about a 50%
decline when cultured for 2–9 days with rhIL-4 (9, 29).
This slow and modest IL-4-dependent decline in Kit levels alone cannot
account for the rapid apoptosis (within hours) observed in the current
study. In our case, the decline in Kit levels observed within the first
2 days of exposure to rhIL-4 and rhSCF was also observed with rhSCF
alone, presumably due to internalization of the Kit-rhSCF complex, as
reported previously (27), an effect from which mast cells
recover as they synthesize new Kit. The dose response of rhIL-4 on cord
blood-derived mast cells examined at 7 days indicated that equal
percentages of tryptase+,
Kit+, and FcRI+ cells
survived at each dose. Thus, rhIL-4-treated cells that survive beyond 2
days are likely to re-express surface Kit.
In contrast to cord blood-derived mast cells used in the current study,
rhIL-4 did not have a substantial effect when added to fetal
liver-derived rhSCF-dependent mast cells after the second week of
culture. However, when rhIL-4 was added to fetal liver progenitors
during the first week of culture, there was a decrease in the numbers
of mast cells along with an induction of surface FcRI expression
(11). Fetal liver-derived mast cells express little, if
any, IL-4R by 4–6 wk of culture, in contrast to the cord blood-derived
mast cells studied herein. Coculture of fetal liver-derived mast cells
with a predominant T cell population derived from CBMCs cultured for
4–5 wk with rhSCF (100 ng/ml) and rhIL-4 (20 ng/ml) did not alter the
fetal liver mast cell response to rhIL-4, making it uncertain whether
the IL-4-mediated apoptotic response is reversible. Also, human
lung-derived mast cells, like those from fetal liver, do not undergo
apoptosis when exposed to rhIL-4. Whether differences between fetal
liver, adult lung, and cord blood-derived mast cells reflect
differences in the tissue source of the progenitors, the maturational
stage of the donor, or the accessory cell populations present at each
site, remain to be understood. Many cells alter their functional
responses to various cytokines as they mature, e.g., the NK cell
response to IL-4 (30). Also, the plasticity of the IL-4
response is unknown. For example, whether the state of fetal
liver-derived and adult lung-derived mast cells can be converted to one
in which rhIL-4 induces apoptosis will be the subject of future
experiments.
Whether the primary pathway leading to rhIL-4-triggered apoptosis in
human cord blood-derived mast cells involved activation of the upstream
caspase-8 or a loss of m was examined
pharmacologically. When upstream activator caspase-8 commits a cell to
apoptosis, the general caspase inhibitor Z-VAD.FMK will prevent both
apoptosis and downstream loss of m
(28). In contrast, when loss of
m commits a cell to apoptosis, leading to
caspase-9 and the caspase-8 activation, Z-VAD.FMK does not prevent the
loss of m, even though apoptosis is
abrogated. The loss of m can be inhibited
in some cells by a combination of CyA and the phospholipase
A2 inhibitor, ArA (24). CyA also
forms a complex with cytosolic cyclophilin A that inhibits calcineurin
(31, 32). However, calcineurin inhibition is not
considered to be involved in preventing loss of
m, because FK506, another inhibitor of
calcineurin, does not prevent the loss of m
(33), as also found in the current study. If
loss of m is the
committing event toward apoptosis, then inhibiting loss of
m will inhibit apoptosis; if activation of
caspase-8 is the primary event, then inhibiting loss of
m will not prevent apoptosis. Experiments
shown in Fig. 7 and Table II favor loss of
m as the event that commits cord
blood-derived mast cells to undergo apoptosis in response to
rhIL-4.
In agreement with previously reported results, cord blood-derived mast
cells expressed both surface FcRI and surface Kit when cultured with
rhSCF alone (2, 34). Our results in the presence of rhSCF
alone differ from reports showing rhIL-4 increases surface FcRI
expression, chymase production, and morphologic maturity of cord
blood-derived mast cells developed in the presence of rhSCF and rhIL-6
(12, 14), and promotes the survival of such CB-MCs at risk
for apoptotic death after withdrawal of rhSCF (7). The
presence of rhIL-6 during the development of CBMC-derived mast cells
may account for the different response to rhIL-4 observed between the
current study and previous ones (12, 13, 14), because rhIL-6
protects cord blood-derived mast cells from rhIL-4-mediated apoptosis.
The mechanism of this protective effect is not yet known, but is under
investigation; for example, surface expression of IL-4R was diminished,
but not eliminated by rhIL-6, even though the cells were protected from
rhIL-4-induced apoptosis. Thus, different culture conditions appear to
produce distinct mast cell responses to IL-4 (35, 36).
The growth, differentiation, recruitment, and activation of cells and
apoptosis of cells often appear to be coregulated. For example, phorbol
esters caused both differentiation and apoptosis of a human myeloid
cell line (37), relating in part to the protein kinase C
isotype repertoire (38), and the expression of a
cyclin-dependent kinase inhibitor (p21) (39). For human
peripheral blood eosinophils, rhIL-4 induces higher cellular levels of
FcRI mRNA at 6 h (40), but apoptosis by 24
h (41). Stromal cell-derived factor-1 (SDF-1), the
chemokine ligand for CXCR4, facilitates both recruitment and apoptosis
of murine and human CD8+ T cells (42, 43). Factors that lead to terminal maturation of a cell often
lead to apoptosis within 5–7 days, whereas those that cause growth
arrest may result in apoptosis developing earlier, typically within 2
days (44). In either case, other factors may oppose
apoptosis. For example, apoptosis of terminally differentiated resting
human neutrophils may occur at sites of inflammation once local
production of sustaining inflammatory mediators wanes
(45). IL-1ß and TNF- are examples of mediators that
protect terminally differentiated human macrophages from apoptosis
(46). In contrast, IL-4 abrogated the protective effect of
IL-1ß on such macrophages, allowing apoptosis to proceed
(47). In that study, treatment of a human myeloblastic
leukemia cell line with TGF-ß1 caused growth arrest and apoptosis
within 2 days, but treatment with IL-6 and TGF-ß1 permitted
differentiation to occur, followed by apoptosis after about 1 wk. IL-6
also protects human myeloma cell lines from apoptosis initiated by
dexamethasone, serum starvation, or Fas (48, 49, 50); rat
pheochromocytoma cells from apoptosis due to serum starvations
(51); human prostate carcinoma cell lines from apoptosis
due to platinum or etoposide (52); and neonatal T cells
from TCR-dependent activation-induced apoptosis (53). IL-4
and IL-7 protect resting murine Th cells from apoptosis
(54), whereas IL-2, IL-4, IL-7, and IL-15 promote the
survival of activated murine T cells both in vivo and in vitro
(55).
Whether the in vitro findings of the current study for human mast cells translate into heterogeneity of the mast cell response to IL-4 in vivo bears considering. Coupling the regulation of apoptosis to differentiation or to activation may provide a pathway to control mast cell numbers in tissues. Production of IL-4 and IL-6 by mast cells, T lymphocytes, eosinophils, and basophils at sites of allergic inflammation is well documented (56, 57, 58, 59, 60, 61, 62, 63). One report indicates that among human mast cells, IL-6 is expressed almost exclusively by the MCT type, the predominant mast cell type in pulmonary tissue, whereas IL-4 is preferentially expressed by the MCTC type (64). Other studies suggest that basophils are far better than mast cells at producing IL-4 (65, 66, 67, 68, 69, 70). We conclude that IL-6 (or a cytokine with comparable activity) at local tissue sites might protect developing mast cells from IL-4-mediated apoptosis and permit accumulation of mast cells at these sites.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Lawrence B. Schwartz, Department of Internal Medicine, Virginia Commonwealth University, P.O. Box 980263, Richmond, VA 23298-0263. E-mail address:
3 Abbreviations used in this paper: SCF, stem cell factor; rh, recombinant human; HMC-1, human mast cell leukemia cell line; CBMC, cord blood mononuclear cells; TRITC, tetramethylrhodamine isothiocyanate; DiOC6(3), 3,3'-dihexyloxacarbocyanine; CyA, cyclosporin A; ArA, aristolochic acid; Z-VAD.FMK, Z-VAD.fluoromethylketone.
Received for publication February 4, 1999. Accepted for publication August 10, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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RI) on human mast cells by IL-4. Int. Immunol. 8:1367.RI): analysis of functional domains of the -subunit with monoclonal antibodies. J. Biol. Chem. 266:11245. RIIb (CD23) expression by IL-4 depends on the stage of maturation of monocytes/macrophages. J. Immunol. 149:1395.[Abstract]
receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc receptor I expression and mediator release. J. Immunol. 162:5455.
stimulates leukemic cell differentiation. Cell Growth Differ. 5:1195.[Abstract]
RI -chain messenger RNA in eosinophils. J. Allergy Clin. Immunol. 96:1161.[Medline]
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