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Faculty of Life Sciences, The Gonda-Goldschmied Center, Bar-Ilan University, Ramat-Gan, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B,
SP increased it and when both were present, SP effect dominated. On the
other hand, the level of IB (NF-B inhibitory molecule) was
decreased by SP, preserved at a relatively high level with HC, and when
both SP and HC were present, SP effect dominated. The intensity of SP
effect, both in vivo and in vitro, its specificity, its inhibition by
SP antagonist, as well as the previously documented presence of SP and
its receptor in the thymus suggest that SP might be a physiological
antagonist of the potent thymocyte apoptosis induced by
GC. |
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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BALB/c male mice, 4–6 wk old, were bred locally or purchased from Tel-Aviv University Animal Research Center (Tel-Aviv, Israel) and were kept under specific pathogen-free conditions. All experimental procedures in animals were conducted according to accepted regulations of animal experimentation under the supervision of our Institutional Animal Care and Use Committee.
Reagents
FCS, penicillin, streptomycin, amphoterycin, L-glutamine, sodium pyruvate, and nonessential amino acids were purchased from Biological Industries (Beth Haemek, Israel). BSA, acrylamide, poly L-lysine, hydrocortisone (HC), propidium iodide (PI), SP, somtostatin, vasoactive intestinal peptide, and [D-Pro2, D-Trp7,9]-SP, [D-Arg1, D-Trp7,9, Leu11]-SP (two SP antagonists (SPA)) were purchased from Sigma (St. Louis, MO).
Antibodies
Fluorescinated anti-mouse CD8 mAb and PE-conjugated
anti-mouse CD4 mAb were obtained from PharMingen (San Diego, CA);
anti-glucocorticoid (GC) receptor (GR) mAb was from ABR-Affinity
Bioreagents (Golden, CO); rabbit anti-SP was from Sigma; rabbit
anti-NF-B p65, and rabbit anti-IB-
(C-21) were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
FITC-conjugated F(ab')2 fragment of goat
anti-rabbit or anti-mouse IgG (H+L), peroxidase-conjugated
affinity-pure goat anti-rabbit, or anti-mouse IgG (H+L) were
obtained from Jackson Immuno-Research (West Grove, PA). FITC-conjugated
anti-leu-4 mAb (anti-human-CD3), from Becton Dickinson
(Mountain View, CA), was used as an irrelevant Ab for flow
cytometry.
Cells and cell cultures
Fresh thymocytes were dissociated from thymuses of BALB/c mice, washed in cold culture medium (RPMI 1640), and exposed to different treatments. The cell lines used in some of the experiments included a murine T cell hybridoma sensitive to HC (2B4.11), kindly provided by Dr. J. D. Ashwell (National Cancer Institute, National Institutes of Health, Bethesda, MD); and a transformed mouse TEC line (27), a kind gift from Dr. A. M. Kruisbeek (Cancer Institute, Amsterdam, The Netherlands). The cells were cultured in tissue culture dishes in RPMI medium for thymocytes and 2B4.11 cells or in DMEM for TEC cells, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml amphoterycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 5 x 10-2 mM 2-ME, and 10% inactivated FCS. The cultures were kept in a 37°C humidified incubator under an atmosphere of 6% CO2 in air.
Analysis of apoptotic death
Apoptosis was induced in thymocytes or 2B4.11 cells by HC (10-6 M), TEC (9), retinoic acid (10-6 M), or gamma irradiation (500 rad). SP (10-9 M) was added at time 0 or later. After 24 h, the cells were centrifuged at 200 x g, the pellet gently resuspended in 1 ml hypotonic fluorochrome solution (50 µg/ml PI, 0.01% sodium azide, 0.1% BSA, 1 mg/ml RNase, and 0.1% Triton X-100 in distilled water) and placed at 4°C in the dark. The stained nuclei were analyzed on a Becton Dickinson FACSCalibur flow cytometer. Apoptotic nuclei were distinguished by their hypodiploid DNA content, compared with the diploid content of normal nuclei.
Flow cytometric analysis of thymocyte subsets
A total of 1 x 106 thymocytes were taken from mice treated by SP and/or HC. Thymocytes were harvested and stained with FITC-conjugated CD4 mAb and PE-conjugated CD8 mAb. Nonspecific staining was assayed with irrelevant mAb (anti-leu-4). Stained cells were analyzed by flow cytometry.
Nucleus/cytoplasm fractionation
A total of 1 x 106 cells were spun in Eppendorf tube and resuspended in 400 µl of buffer A (10 mM HEPES, 10 mM KCl, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine, 1 mM Na metabisulfite, 0.1 mM EDTA, 10 µg/ml leupeptin, 50 µg/ml aprotinin, and 1 µg/ml pepstatin). The cells were allowed to swell on ice for 15 min, and then 25 µl Nonidet P-40 (10%) was added. The tube was vortex mixed (10 s) and centrifuged (30 s). Supernatant (cytoplasmic fraction) was collected, and the pellet (nuclear fraction) was resuspended gently in 50 µl ice cold buffer C (same as buffer A with the exception of 0.4 M NaCl instead of 10 mM KCl and 500 µg instead of 50 µg aprotinin). The tube was rocked at 4°C for 15 min and spun in the cold for 10 min.
Immunostaining
For detection of SP in the thymus, cryosections of mouse thymus
were fixed with methanol/acetone (1:1), incubated with rabbit
anti-SP Abs, and then with FITC-labeled goat anti-rabbit IgG
Abs. Detection of SP receptor on isolated washed thymocytes was
accomplished by first incubating methanol/acetone fixed cells with SP,
then proceeding as for SP detection. Staining for GR or NF-B was
done on thymocytes or 2B4.11 cells, which were adhered on poly
L-lysine-coated slides, fixed with methanol/acetone (1:1),
treated with 0.01% Triton X-100, and then incubated with the
respective first and second Abs (see "Antibodies"). After
incubation, the slides were washed, mounted, and examined under a
fluorescence microscope or confocal microscope (MRC 1024; Bio-Rad,
Richmond, CA) (28).
Immunoblotting
Cells were washed in PBS and lysed in lysis buffer (25 mM Tris base (pH 7.4), 50 mM NaCl, 0.5% Na-deoxycholate, 2% Nonidet P-40, 0.2% SDS, and 1 mM PMSF). The proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with milk and probed for 1 h with the first Ab, washed in PBS, and detected using a peroxidase-conjugated second Ab and its chemiluminescent substrate (Pierce, Rockford, IL) (9).
Statistical analysis
Each experiment was repeated three to five times. The pooled data were statistically analyzed by the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first addressed the question: could SP affect stress-induced
deletion of thymocytes? To this end we selected HC as the main mediator
of stress-induced thymus depletion. HC either alone or with SP was
injected into adult mice (i.p). After 72 h thymocytes were counted
and thymocyte subsets were determined by flow cytometry. As expected,
HC induced a marked reduction in observed thymus size, which was
accompanied by a 78% reduction in the number of thymocytes (Fig. 1
A). This reduction was
completely prevented by injecting SP together with HC. Flow cytometric
analysis demonstrated (Fig. 1B) that the loss of cells by HC
was mainly in the DP thymocyte subpopulation (90% reduction, as
calculated from the percentage of DP cells (Fig. 1B) and
total thymocyte counts (Fig. 1A)). SP completely abolished
HC effect; it prevented the loss of thymocytes by rescuing the DP
thymocytes from HC-induced death and not by increasing other thymocyte
subpopulations (Fig. 1B). Addition of SPA
([D-Arg1, D-Trp7,9,
Leu1] SP or [D-Pro2,
D-Trp7,9] SP) blocked the effect of SP on
thymocyte number (Fig. 1A) and subsets (Fig. 1B),
suggesting that SP effect is a receptor-mediated effect. Injection of
SP or SPA alone did not change either the total number of viable
thymocytes nor the proportion of different thymocyte subsets.
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The in vivo experiments left a basic question unanswered: is SP
effect a direct one on thymocytes, or indirect via induction of other
in vivo regulatory loops. Indeed, we confirmed previous reports
(18, 19, 20) on the presence of SP in the thymus and SP
binding sites on thymocytes (data not shown), suggesting that SP effect
should conceivably be a direct one. However, we looked for a more
conclusive answer to this question in vitro. Freshly isolated
thymocytes from untreated mice were incubated in culture with HC for
24 h in the presence or absence of SP and analyzed for apoptosis
by flow cytometry after nuclear staining with PI. Indeed, 37% of the
thymocytes died within 24 h in culture medium; HC increased this
percentage to 84%, and when both SP and HC were present, the
percentage of apoptotic cells was the same as in the control (Fig. 2). Furthermore, addition of SPA to the
culture inhibited the SP effect. Both SPAs had similar activity in
neutralizing SP effect. Therefore, in subsequent experiments we used
only one of the antagonists ([D-Arg1,
D-Trp7,9, Leu11] SP). SP
or SPA alone had no effect on thymocyte viability (Fig. 2). Thus, SP
has a direct, receptor-mediated, antagonistic effect in preventing
HC-induced thymocyte apoptosis.
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The effect of SP on thymocyte apoptosis induced by different means
was assayed. SP did not have any effect on thymocyte apoptosis
induced by irradiation, by retinoic acid, or by TEC (Table I), suggesting that SP blocks a specific
HC induction step of cell death programming and not a step in the
common final pathway of thymocyte apoptosis. Furthermore, two other
neuropeptides, somatostatin and vasoactive intestinal peptide, which
are known to be produced and secreted in lymphoid organs, had no effect
on HC-induced thymocyte apoptosis (Table II). These results indicate that the
effect demonstrated here is specific to both HC and SP.
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The time at which SP can be added to HC containing thymocyte
culture and still have a neutralizing effect on HC may give a clue as
to the mechanism of effect. For these and subsequent experiments, we
used the hybridoma cell line 2B4.11, which is sensitive to GC
(8) and has very low background apoptosis (2–5% at
24 h). Incubation of 2B4.11 cells with HC resulted in 30–50%
apoptosis (Fig. 3), an effect that could
be neutralized by SP. As before, SP effect was antagonized by SPA (Fig. 3). In subsequent experiments with 2B4.11 cells, SP was added at
various times after HC (1–5 h). The results indicate (Fig. 4) that at 1-h delay, SP was fully
effective; at 2 h SP had only a partial effect, which disappeared
completely at a 3-h delay. These results suggest that the effect of SP
on HC-induced apoptosis should be looked for in events triggered by HC
within the first 2 h.
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The fact that SP inhibits HC-induced apoptosis specifically and
only when added at time 0–2 h suggests that SP exercises its
inhibitory effect on an HC-specific early event. The central HC-related
early event is HC binding to its cytoplasmic receptor (GR) in the
cytoplasm, and translocation of the GR-HC complex to the nucleus
(29, 30). Analysis of GR expression was performed on
2B4.11 cells by incubating the cells for 0–4 h with SP or HC or both
and then extracting their cytoplasmic and nuclear fractions. Immunoblot
analysis of GR expression indicated that GR level in cytoplasm (Fig. 5A) rises within 1 h
after incubation with HC (densitometry (D); all D values are relative
to control = 1.00; HC at 1 h = 3.06), peaks at 2 h (D =
6.18), and then declines; at 4 h GR levels were still higher than
in the control (D = 1.87). A similar rise in GR level was observed
in nuclear extract (Fig. 5B), with a peak sustained until
the third hour (D = 3.73), and a decline at 4 h (D
= 1.94), which did not reach yet control level. SP by itself had no
effect on GR expression, but strongly inhibited the HC-induced rise in
GR levels in both the cytoplasm (D at 1–4 h = 0.29–0.78) and the
nucleus (D at 1–4 h = 0.7–1.95) (Fig. 5, A and
B).
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). In the control, GR is virtually
undetectable (Fig. 6A); it is strongly stained in both the
cytoplasm and the nucleus after 3 h incubation with HC (Fig. 6B). SP completely abolished detectable GR in the cytoplasm,
which appeared as the control. However, in the nucleus, GR could be
detected, in the cells treated by SP + HC, albeit at a clearly lower
level than in HC-treated cells (Fig. 6C). The addition of
SPA to the SP + HC incubation mixture resulted in GR appearance in both
the cytoplasm and nucleus at about the same level as with HC alone.
However, staining pattern was somewhat different: spotty with HC, and
more diffuse with HC + SP + SPA (Fig. 6D). The significance
of this difference is not clear at the moment. In conclusion, both
immunoblotting and confocal microscopy clearly demonstrated strong and
specific inhibitory effect of SP on GR production.
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B/IB system in HC/SP interaction
The NF-B/IB system, which is known to be modulated by both
HC and SP (31, 32) and to be involved in apoptosis
induction (33), was an obvious candidate for analysis in
our model. Indeed, immunoblot analysis of 2B4.11 nuclear extracts (Fig. 7) indicated that SP by itself increased
NF-B expression (D = 1.00, 1.60, 2.43, and 2.69, at 0, 1, 2,
and 3 h, respectively). HC decreased it (D = 0.18 and 0.43 at
1 and 2 h, respectively) and when both were present, SP effect
dominated (D = 3.09 and 1.96 at 1 and 2 h, respectively).
These results were confirmed by staining for NF-B (counterstain, PI)
and confocal microscopy (Fig. 8); SP
enhanced NF-B expression and translocation into the nucleus (Fig. 8B). HC slightly inhibited cytoplasmic expression and
completely abolished nuclear translocation of NF-B (Fig. 8C). In the presence of both HC and SP, the SP effect
dominated (Fig. 8D).
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B changes by immunoblot (Fig. 9) suggested that it is also involved in
HC/SP antagonism; SP caused a progressive decrease in IB levels
(D = 1.00, 0.81, and 0.45 at 0, 2, and 3 h, respectively), HC
preserved it at a relatively high level (D = 1.23 and 1.29 at 2
and 3 h, respectively), and in the presence of both HC and SP, SP
effect dominated (D = 0.78 and 0.48 at the same times).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first key event is GR expression and translocation into the nucleus. GR is an intracellular receptor present in the cytoplasm in a complex which keeps it stable and inactive (34, 35, 36). When a specific ligand (HC or a semi-synthetic GC) enters the cell, the receptor is activated, passes into the nucleus (translocation), and binds to a specific regulatory DNA site in several genes. As a result, some of these genes will be transactivated and some will be transrepressed as a function of cell type, developmental stage, physiological state, and presence of other transcription factors (34, 35, 37, 38). The same gene may sometimes be transactivated and sometimes transrepressed. The GR gene itself is a good example. Activated GR autoregulates its own level (39, 40, 41), usually down-regulation (transrepression) (42), which is needed to temporally limit and attenuate the potent GC-induced physiological changes. However, up-regulation of GR production by GR (transactivation) was also reported in several cases, e.g., T but not B cell lines (39, 43, 44), either constantly or as an initial phase (biphasic regulation) (42). Such an amplified response may be required when the cell undergoes a profound physiological change, such as apoptosis induction (39).
We demonstrated a strong up-regulation of GR level induced by HC in
addition to the process of GR translocation. These simultaneous
processes were clearly seen both in the immunoblot (Fig. 5
) and by GR
staining and confocal microscopy (Fig. 6B). The increase in
GR expression in our system reached a peak at 2 h, quicker than
reported in other systems (39). The difference may be
attributed to the different cell systems; thymocytes are most sensitive
to GC effect. It may be interesting to comparatively study the
mechanisms regulating GR levels in different cell systems
(39, 40, 41, 42, 43, 44). SP had reduced GR levels in our system in a
strong, specific, and consistent manner. The question still remains
whether SP effect on the GR level is central for apoptosis inhibition.
SP manifests a complete inhibitory effect when added to the cells
1 h after HC (Fig. 4), at a time when GR translocation (and DNA
binding) takes place already (29, 30). Indeed, the final,
no-return event in apoptosis induction by HC may be later than GR-DNA
binding (7, 45), thus allowing SP to exert its effect on
the process. Although this possibility may be further explored, a role
for NF-B, as an internal mediator of SP effect, looks more
attractive for the following reasons.
NF-B is a key transcription factor in immune and inflammatory
processes (46, 47, 48, 49). It is also a central player in
apoptosis, involved in its induction in some cell systems
(50) and in protection from it in many other systems
(51). In lymphoid cells, NF-B usually serves an
anti-apoptotic function (52). Out of several forms of
NF-B, the one containing RelA (p65) is most relevant for apoptosis
inhibition (53). There are certain similarities between
NF-B and GR as transcription factors. Both are present
constitutively in the cytoplasm in inactivating complex with proteins:
heat shock and other proteins in the case of GR (30, 36),
and IB in the case of NF-B (54). Upon specific
activation they translocate into the nucleus and bind to their
respective specific regulatory DNA segments. Furthermore, although
these two factors are mostly antagonistic in their function, there is a
meaningful cross-talk between them and, depending on the specific
condition, one dominates over the other. GR inhibits NF-B-mediated
inflammatory responses, whereas a rise in NF-B level inhibits
GR-mediated gene transactivation (31). This cross-talk
takes place by direct physical interaction of the two factors
(55, 56, 57). Indeed, GC induce the synthesis of a
NF-B-specific inhibitor, IB (58). However, it was
questioned to what extent this effect is physiologically meaningful in
controlling NF-B activity (59). Finally, SP was
recently found to be a potent inducer of NF-B activity
(32), an effective reasonable association of extra- and
intracellular mediators of inflammation. Indeed, the fact that the
optimal concentration of SP
(10-8–10-12 M) is much
lower than that of HC
(10-5–10-7 M) (our work
and Refs. 60 and 61) suggests that NF-B
amplifies the SP effect in the cells. All these data suggest that
NF-B should be most relevant in our system.
Indeed, SP induced an impressive increase in the NF-B level, HC
caused modest a decrease in it, and when both agents were present, the
SP effect dominated. SP-induced NF-B activation is a quick process:
a substantial increase was observed within 1 h (Fig. 7), actually
in 30 min (32) and maybe even less, leaving enough time
for manifesting the anti-apoptotic effect of NF-B, even when SP
addition is delayed for 1 h. The conditions which allow NF-B
dominance over GR are not yet clear. One possibility is combined
NF-B/AP-1 activation, which probably takes place by SP (32, 62). One may speculate that a synergistic effect of these two
factors may overcome the GC effect. It should be mentioned that both
NF-B and AP-1 interact physically with GR (38, 56). It
will be interesting to test the involvement of AP-1 and other relevant
transcription factors in the SP/HC interaction and to employ
pharmacological and molecular inhibitors of NF-B (63)
to further analyze the role of NF-B in the process. Finally, IB
is also modified by SP and HC, with SP dominating in the interaction.
It has to be further elucidated how much it contributes to the
modulation of NF-B activity, in view of the conflicting reports on
the subject (58, 59).
In the present study we demonstrated that SP acts strongly and
efficiently against one of the most powerful and impressive activities
of GC: apoptosis induction in DP thymocytes. The intensity of SP
effect, both in vitro and in vivo, its specificity, as well as the
presence of SP and its receptor in the thymus suggest that we are
dealing with a fundamental physiological regulatory tool. Apoptosis is
a key event in metazoa, and therefore it should be strictly controlled,
both at the intracellular level and at the level of the whole organism.
As in several other critical body systems, in which hyper- or
hypofunction may endanger existence, precise regulation is achieved by
multilevel, multifactorial control mechanisms, which complement or
antagonize each other. The use of antagonistic mechanisms is vital to
achieve fine tuning of the desired response. The present study suggests
that SP is a most efficient antagonist of HC in the process of
apoptosis induction. The two intracellular events analyzed here,
changes in GR and NF-B, should be regarded as representatives of a
complex process, by which the antagonistic signals of SP and HC coming
from the external milieu of the cell may be deciphered and direct
cellular "decisions" about life and death.
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Jacob Shoham, Faculty of Life Sciences, The Gonda-Goldschmied Center, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail address:
3 Abbreviations used in this paper: DP, double positive (CD4+CD8+ T cells); D, densitometry; GC, glucocorticoid; GR, GC receptor; HC, hydrocortisone; PI, propidium iodide; SP, substance P; SPA, SP antagonist; TEC, thymic epithelial cell.
Received for publication May 18, 1999. Accepted for publication December 14, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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