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Departments of
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Medicine and
Cell Biology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiation (dependent on
IL-1 converting enzyme family proteases), ceramide, and mitogen
challenge, suggesting functional integrity of the apoptotic pathway.
Furthermore, the defect in Fas-dependent apoptosis was overcome by
prestimulation with allogeneic accessory cells instead of mitogen.
Taken together, the findings suggest that selective resistance to
Fas-dependent apoptosis reflects altered Ag-driven, accessory
cell-dependent signaling and that ineffective activation of Fas signal
transduction may contribute to T cell-dependent immunoinflammation in
asthma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy nonasthmatic control subjects (6 male and 4 female, aged 20–54 yr) and subjects with asthma (3 male and 14 female, aged 17–64 yr) were recruited using informed consent for a protocol approved by the University Committee for Human Research. Nonasthmatic subjects had no clinical history of airway obstruction, normal forced expiratory volume in 1 s (FEV1)3 (81–129% predicted), and normal airway reactivity to inhaled methacholine (FEV1 PC20 > 16 mg/ml). Asthmatic subjects had a clinical history consistent with intermittent and reversible airway obstruction, mean FEV1 of 85% predicted (range, 42–114%), and hyperreactivity to inhaled methacholine (FEV1 PC20 = 1.51 ± 0.41 mg/ml; range, 0.06–5.40 mg/ml). Two subjects were being treated with inhaled and oral glucocorticoids (GCs) at the time of study. Positive skin test reactivity to a panel of allergens (house dust, trees, grasses, fungi, and dog and cat dander) was present in 14 of the asthmatic subjects and 5 of the control subjects. For GC-withdrawal experiments, six asthmatic subjects were treated with inhaled triamcinolone (1600 µg/day for 30 days) before the first assessment of T cell function. Triamcinolone treatment was discontinued and subjects were monitored for an additional 6 wk or until peak expiratory flow had decreased by 20% at which time a second assessment of T cell function was obtained.
T cell culture
PBMCs were isolated using Ficoll-Hypaque (Pharmacia,
Piscataway, NJ), and placed at 5 x 106 cells/ml/10-mm
well in RPMI 1640 supplemented with 7.5% FBS, L-glutamine,
nonessential amino acids, sodium pyruvate, 2-ME, and
penicillin/streptomycin. Isolated cells exhibited 100% viability by
trypan blue exclusion and no detectable contamination by granulocytes
or erythrocytes. The protein form of PHA (PHA-P) (10 µg/ml)
was added to the medium on day 0, and IL-2 (50 U/ml) was added on day 1
and then every 3 days. Cell cultures were maintained at a density of
1–2 x 106 cells/ml and were harvested at weekly
intervals, each 3 days after the last addition of IL-2. For allogeneic
stimulation, PBMCs (5 x 106/ml) were stimulated with
-irradiated (20 Gy) allogeneic PBMCs (1 x 106
cells) from other nonasthmatic or asthmatic subjects. The next day cell
cultures were diluted with medium (1:1, v/v) containing IL-2 and
maintained for 2 wk as described above.
Fas activation
T cells were placed into 10-mm wells (3 x 105 cells/0.5 ml/well) with medium alone or with media containing 2 µg/ml of mouse anti-Fas IgM-mAb CH-11 (Upstate Biotechnology, Lake Placid, NY) or IgG1-mAb DX-2 (PharMingen, San Diego, CA) as well as control IgM- or IgG1-mAb for 18 h at 37°C. For IgG1 mAbs, wells were precoated with goat anti-mouse IgG.
Apoptosis assays
For flow cytometry, cell samples were resuspended in PBS containing 50 µg/ml propidium iodide (PI; Boeringer Mannheim, Indianapolis, IN) for 15 min to achieve maximal staining and then were analyzed for cell size (forward-angle light scatter), density (side-angle light scatter), and membrane integrity (PI exclusion) using an Epics Elite (Coulter, Hialeah, FL) or FACSCalibur (Becton Dickinson, Mountain View, CA) flow cytometer as described previously (12, 13). PI staining vs forward-angle light scatter was used to calculate the percentage of live cells in each sample based on 30,000 events. Phosphatidylserine externalization was detected by labeling cells with 1-palmitoyl-[6-[(7-nitro-2–1, 3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoserine (NBD-PS; Avanti Polar Lipids, Alabaster, AL) and monitoring the level of this fluorescent phospholipid analogue on the cell surface by flow cytometry as described previously (14). To assess nuclear morphology, 1 x 105 cells were spun onto a microscope slide, fixed in methanol, and incubated with PBS containing Hoechst dye no. 33342 (10 µg/ml, Molecular Probes, Eugene, OR) for 10 min at 25°C. Slides were rinsed in PBS and mounted in Vectashield (Vector Laboratories, Burlingame, CA) for fluorescence microscopy. To assess DNA fragmentation, cells (2 x 106 cells/condition) were lysed, and the DNA was subjected to electrophoresis in a 1.8% agarose gel containing ethidium bromide as described previously (15).
T cell phenotyping
Levels of Fas and other cell surface proteins were determined by flow cytometry using T cells cultured from nonasthmatic and asthmatic subjects. For Fas levels, cell samples (2 x 105 cells/condition) were incubated with anti-Fas mAb DX-2 (2 µg) followed by FITC-labeled goat anti-mouse Ab (1:150) or phycoerythrin (PE)-conjugated anti-Fas mAb (Becton Dickinson) in PBS containing 0.2% BSA and 0.01% sodium azide for 20 min at 4°C. For other T cell markers, cells were labeled with mAbs to CD2, CD3, CD4, CD8, CD25, CD45RO, or HLA-DR either as FITC- or PE-conjugated or as unconjugated followed by secondary labeled Ab (Becton Dickinson). Labeled cells were fixed in 1% paraformaldehyde and analyzed by flow cytometry to determine the percentage of positive cells in each sample based on 10,000 events in live cell gate/condition and corrected for background detected with isotype-matched control for primary mAb.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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a and b). By contrast, T cell cultures from
asthmatic subjects exhibited an invariant defect in Fas-dependent
apoptosis throughout the culture period of 4 wk (Fig. 1, a
and b).
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c). Taken together, these data
indicate that the observed resistance to Fas-induced cell death is a
stable phenotype in cultures of T cells from asthmatics and is not
dependent on concomitant treatment with GCs. Initial data also
indicated that Fas resistance did not appear to depend on atopy
(because atopic and nonatopic subjects were present in nonasthmatic and
asthmatic groups). Thus, we detected Fas resistance in three of three
nonatopic asthmatic subjects. However, a larger sample size will be
required to be certain that Fas resistance in asthma is not dependent
on atopy.
Fas resistance in asthma was also observed when apoptosis was
determined by additional assays of apoptosis. Thus, assays of
phosphatidylserine externalization (using a phospholipid analogue and
flow cytometry), nuclear morphology (using a DNA-binding dye and
fluorescence microscopy), and DNA degradation (using gel
electrophoresis), each confirmed that Fas-dependent T cell apoptosis
was decreased in T cells from asthmatic vs control subjects (Fig. 2 and data not shown).
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and data not shown). In particular,
asthmatic subjects with normal levels of Fas still exhibited a marked
decrease in Fas-dependent apoptosis. Asthmatic subjects had 23% lower
levels of Fas+ T cells during treatment with GCs, but this
treatment (as noted above) was also unrelated to Fas resistance. The
poor correlation between Fas-dependent apoptosis with Fas levels is
consistent with the observation that Fas levels are not predictive of
biologic responsiveness (18).
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).
In particular, we found the usually low percentage of
CD4-CD8- T cells in asthmatic and
nonasthmatic subjects in freshly isolated (19), and cultured T cells
(Fig. 3), in contrast to the increase in double-negative T cells found
in association with Fas mutations (10, 11).
The observed defect in T cell apoptosis appeared independent of
mitogen-stimulated T cell proliferation and specific for the
Fas-dependent pathway. Thus, initial PHA stimulation causes equivalent
proliferative responses in T cell cultures from asthmatic and
nonasthmatic subjects (data not shown). In addition, other inducers of
apoptosis such as -irradiation caused the same degree of apoptosis
in T cells cultured from asthmatic and nonasthmatic control subjects
(Fig. 4). Apoptosis induced by
-irradiation (and Fas activation) involves sequential activation of
members of the IL-1 converting enzyme (ICE) superfamily of proteases
known as caspases (20). Taking advantage of this commonality in
signal transduction and the use of peptide-fluoromethylketone (FMK)
reagents as probes for ICE family activity (21), we found that the
apoptotic response to -irradiation was blocked almost completely by
a cell-permeable synthetic peptide designed to inhibit ICE
(z-VAD-FMK) and only partially by an inhibitor of
CPP32 activity (z-DEVD-FMK) in both asthmatic and nonasthmatic
subjects. These findings are consistent with a distinct requirement for
ICE family proteases for -irradiation-induced apoptosis in human T
cells and one that is unchanged in asthma.
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-irradiation) have been linked to sphingomyelinase-dependent
generation of ceramide (22). In a further comparison of T cell
apoptosis in asthmatic vs nonasthmatic subjects, we found that ceramide
treatment resulted in a similar degree of apoptosis for T cells taken
from both groups of subjects (Fig. 4). Concentration-response
relationships for ceramide-induced apoptosis (using 1–100 µM of
C2-ceramide) were also similar for T cells taken from asthmatic and
control subjects (data not shown). Ceramide-induced apoptosis was not
prevented by z-VAD-FMK or z-DEVD-FMK (Fig. 4), consistent with
observations by others (23) and us (data not shown) using T cell lines.
Taken together, Fas resistance in asthmatic T cells did not appear to
be due to T cell defects in Fas expression, caspase activities, or
ceramide-driven events and so more likely may represent a defect
in initial triggers leading to Fas activation.
Accordingly, we tested whether initial mitogen stimulation of T cells
(by PHA in the presence of accessory cells) and the consequent defect
in Fas-triggered apoptosis indicated an underlying defect in accessory
cell action. Initial evidence of this possibility was obtained when
rechallenge with PHA (2 wk later when accessory cells were depleted;
3 caused equivalent apoptosis in T cells cultured from asthmatic
and nonasthmatic subjects (Fig. 5a). More direct evidence was
provided by experiments using -irradiated allogeneic PBMCs in place
of PHA for initial T cell stimulation. In this case, we observed
reversal of the expected resistance in Fas-dependent apoptosis in
asthmatic subjects (Fig. 5b) and an apoptotic response
similar to normal subjects (data not shown). Phenotype analysis of T
cell cultures indicated a slight increase in Fas and a marked increase
in IL-2R (CD25) expression for allogeneic over PHA stimulation (Fig. 5c), consistent with greater potency of nonself over
mitogenic stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiation) act normally; 2) ICE activation and ceramide action
appear normal; 3) and the defect is overcome by T cell stimulation with
allogeneic accessory cells. Based on these observations, Fas resistance
in asthmatic subjects may be due to altered T cell or accessory cell
capacity for activation of Fas-dependent events. Thus, subtle T cell
alterations in Fas function, signaling (by Daxx- or
FADD-dependent pathways (24)), or dampening (by
Bcl-xL (25), FAP-1 (26), sentrin (27), viral (28) or
nonviral (29, 30) FLIPs) as well as a defect in accessory cell
capacity to activate Fas-dependent events may underlie the observed Fas
resistance in asthmatic subjects. Other deficiencies in accessory cell
function (e.g., decreased IL-12 production; 31 may support a more
general but distinct defect in accessory cell regulation of T cell
function in asthma. In that context, asthma may share a propensity for
decreased IL-12 production and a Th2-cytokine profile (32) but does not
manifest preferential expansion of CD4-CD8- T
cells or autoimmunity, all abnormalities that are characteristic of
genetic defects in Fas or Fas ligand (1, 10, 11). We do not yet have
direct evidence that resistance to Fas-mediated apoptosis in cultured T
cells precisely reflects the T cell behavior in vivo. However, any
defect that limits T cell apoptosis in asthma could serve to
selectively amplify immune cell accumulation at an airway site in which
Ag-specific T cells are then rendered competent for unrestricted
autocrine and paracrine activities. The manifestation of the defect in
the airway likely relates to this site as one that is commonly exposed
to allergen and even normally operates at a lower level of efficiency
to protect against allergic sensitization (33).
In addition to Fas-induced apoptosis, we note that T cell cultures from
normal control and asthmatic subjects exhibit a significant basal level
of cell death (based on PI staining). At least some of this basal
effect is due to nonspecificity of PI staining. These characteristics
of T cell culture and PI staining lead us and others to use a
definition of specific cell death based on the absence of PI staining
and normal forward-angle light scatter as well as the analysis of data
for percentage increases in cell death above background levels (as
noted above and in 12 . The basis for basal cell death during
culture is uncertain, but there is no evidence that it is mediated by
the Fas system. Thus, Fas-mediated death normally requires expression
of Fas ligand, and ligand is nearly undetectable under these control
culture conditions (see Fig. 5).
The precise molecular basis for resistance to Fas-mediated apoptosis in asthma remains uncertain, but our results address several possibilities. Thus, as noted above, it appears that decreased levels of Fas receptor do not account for resistance, but an alteration in receptor signaling remains a possible mechanism. Other work suggests that Fas signaling may differ when triggered by anti-Fas mAb vs the different forms of Fas ligand (34). In that regard, our initial experiments indicate the same profile of apoptotic responsiveness with soluble Fas ligand as with anti-Fas mAb in T cell cultures from nonasthmatic and asthmatic subjects, (S.J. and M.J.H., unpublished observations). However, it is possible that membrane and soluble forms of Fas ligand may exert differential effects on Fas signaling in peripheral blood T cells (34), and this possibility as well as other features of the Fas receptor complex still need to be defined.
Nonetheless, the capacity of Fas ligand to trigger T cell death implies a role for the Fas/Fas ligand system in controlling the endogenous T cell response to allergen stimulation. In the context of the asthma, it would therefore be useful to analyze Fas-mediated apoptosis in response to naturally occurring allergens in addition to the present use of more generic T cell activation by PHA or alloantigen. An assessment of allergen-induced apoptosis would require isolation of allergen-specific T cell clones from asthmatic subjects as well as from a control group without asthma, such as those with allergic rhinitis. Our initial results suggest that resistance to Fas-mediated apoptosis may be lost during long-term culture and repeated stimulation required for T cell cloning (S.J. and M.J.H., unpublished observations), but additional work will be needed to more fully address this question. In that same context, we also note that asthma is characterized by a relative increase in CD4+ T cells with a Th2-type cytokine profile (6). More recent work with highly polarized CD4+ T cell clones, derived from transgenic mice, provides evidence of preferential Fas-mediated Ag-stimulated apoptosis in Th1 vs Th2 effector subsets (35). Thus, a skewed Th2-type profile in asthma may also reflect the observed defect in T cell apoptosis because T cells from asthmatics are in an environment in which the Fas system is less active and so may preferentially eliminate the more susceptible Th1 cells. As was the case for allergen stimulation experiments, preferential sensitivity of Th subsets may also be best addressed by generating T cell clones with a homogenous Th1 or Th2 cell phenotype.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael J. Holtzman, Washington University School of Medicine, Campus Box 8052, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address:
3 Abbreviations used in this paper: FEV1, forced expiratory volume in 1 s; FEV1 PC20, provocative concentration for 20% increase in FEV1; GC, glucocorticoid; PI, propidium iodide; PE, phycoerythrin; FMK, fluoromethylketone; NBD-PS, 1-palmitoyl-[6-[(7-nitro-2–1, 3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoserine; ICE, IL-1 converting enzyme.
Received for publication July 10, 1998. Accepted for publication October 10, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1+ T cell adherence and tissue retention. J. Immunol. 155:1117.[Abstract]
release in patients with allergic asthma. J. Immunol. 158:5560.[Abstract]
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