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J Biol Chem, Vol. 273, Issue 44, 28897-28905, October 30, 1998
From the Departments of A new quantitative cytometric technique, termed
the ArrayScanTM, is described and used to measure NF- IL-11 and TNF Cellular assays of the early signaling events leading up to NF- Cells--
Human THC-igfI chondrocyte cells were obtained from
M. B. Goldring (27) and passaged weekly in Dulbecco's modified
Eagle's medium (low glucose; Life Technologies, Inc.) with 10% FBS.
HeLa cells were obtained from the ATCC collection and passaged weekly in Dulbecco's modified Eagle's medium with high glucose. For
experiments, cells were removed with 0.25% trypsin/EDTA, plated in
96-well plates (Polyfiltronics, Rockland, MA) at 10,000 cells/75
µl/well, and grown for 20 h.
Translocation Experiments--
The plates were washed once in
Dulbecco's modified Eagle's medium without FBS, and 90 µl of fresh
medium was added. Various compounds were added in Me2SO,
and up to 1% Me2SO could be added without any effects on
NF- Cell Fixation and Staining--
The cells were fixed with 100 µl of 4% formaldehyde in phosphate-buffered saline for 20 min at
room temperature, permeabilized with 100 µl of 0.1% Triton X-100 in
phosphate-buffered saline for 5 min at room temperature, and then
washed twice with 300 µl of 0.1 M Tris-HCl buffer, pH
7.8. To block nonspecific antigenic sites, the wells were incubated for
20 min with 100 µl of 5% nonfat dry milk in 0.1 M
phosphate buffer, pH 7.8, at room temperature. After washing two times
in 0.1 M Tris wash buffer, the cells were incubated for
1 h with 100 µl of rabbit anti-p65 NF- Data Acquisition and Analysis--
The ArrayScanTM cytometer
(Cellomics, Inc., Pittsburgh, PA) is an automated fluorescent imaging
microscope for extracting information about the spatial and temporal
distribution of fluorescently labeled components in cells grown in the
microtiter plates (26). The system was used to scan multiple fields
from well to well and to acquire and analyze each of the cells in the
images according to the algorithm described in Fig. 2. Within each
well, multiple cellular images/well were acquired by moving the
position of the plate the width of one image field (350 µm) in a
square pattern of locations centered on the center of the well. In each
well, images were acquired until a preselected number of cells had been imaged and analyzed. The ArrayScanTM system consists of an optical system with a spatial resolution of 0.68 µm (Carl Zeiss, Inc., Thornwood, NY); a triple band fluorescence emission filter set with
matched single band excitation filters for selectively imaging Hoechst,
FITC, or Texas Red (model XF57, Omega Optical, Brattleboro, VT); a CCD
camera with frame grabber; and a Pentium PC computer and applications
software.
Immunoblotting and Densitometry--
Cells grown on 96-well
plates were lysed in 30 µl of 2× SDS-PAGE sample buffer, combining
eight wells into one sample (2.4 × 105 cells).
Alternatively, cells were grown in 35-mm clusters at the same density
(106 cells/well) and solubilized in 50-µl sample buffer.
SDS-PAGE was performed on 10% Novex gels (San Diego, CA) using
6.4 × 104 cell equivalents/lane. Immunoblotting was
performed with rabbit anti-I Cytometric Measurements of p65 NF-
Quantitation of the amount of p65 found in each cell is shown in 16 representative unstimulated and IL-1-stimulated HeLa cells (Fig.
5). The amount of p65 fluorescence found
in the nucleus is a composite of background fluorescence plus a small
amount of cytoplasm surrounding the nucleus, the result averaged across all of the pixels within the nuclear area (see Fig. 2, Side
View). As is seen visually in Figs. 1, 3, and 4, the calculated
p65 nuclear fluorescence increases after stimulation (Fig.
5A). The cytoplasmic fluorescence is a composite of
background fluorescence within the cell as well as some background
fluorescence observed in the annular ring that might fall outside of
the cells (see Fig. 4). The occasional placement of the cytoplasmic
ring in part outside of the cells often led to a reduced mean
cytoplasmic intensity because of averaging in noncytoplasmic regions.
After stimulation, the cytoplasmic staining was only partially reduced,
suggesting only a partial translocation of NF- Time Course of NF-
Because NF- IL-1 and TNF Cell Numbers Required for Statistical Significance--
To
determine the minimal number of cells required for the assay to assure
statistical significance, a series of plates containing unstimulated
and IL-1 Assay Variability--
Several replicate plates of chondrocytes
stimulated with IL-1 Effects of Cytokine and Receptor Antagonists on NF-
A similar inhibition was performed with antibodies specific for
individual cytokine receptors. IL-1 stimulation of chrondrocyte or HeLa
cell NF- Effect of Proteasome and Kinase Inhibitors on NF-
The nonspecific kinase inhibitor K252b has been shown to inhibit the
phosphorylation of both the IL-1 receptor association kinase and
I In the present paper, we describe a new technique for quantitation
of early events in IL-1 and TNF Previously, NF- As is shown here, IL-1- or TNF Both IL-1 and TNF In this paper, we discuss the effects of inhibitors of NF- *
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
IL, interleukin; IL-1RA, IL-1 receptor antagonist; TNF, tumor necrosis factor; TNFR, TNF
receptor; PAGE, polyacrylamide gel electrophoresis; LLL-H, Leu-Leu-Leu
aldehyde.
2
D. K. Miller and S. M. Raju,
unpublished observations.
Characterization and Quantitation of NF-
B Nuclear
Translocation Induced by Interleukin-1 and Tumor Necrosis
Factor-
DEVELOPMENT AND USE OF A HIGH CAPACITY FLUORESCENCE CYTOMETRIC
SYSTEM*
,,,,
Immunology and Inflammation
and § Biometrics Research, Merck Research Laboratories,
Rahway, New Jersey 07065 and ¶ Cellomics, Inc.,
Pittsburgh, Pennsylvania 15238
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B nuclear
translocation induced by interleukin (IL)-1 and tumor necrosis
factor-
(TNF). The amount of p65 staining is measured in both the
nuclei defined by Hoechst 33342 labeling and in the surrounding
cytoplasmic area within a preselected number of cells/well in 96-well
plates. Using this technique in synchronously activated human
chondrocytes or HeLa cells, NF-B was found to move to the nucleus
with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes,
a rate in each case about 4-5 min slower than that of IB
degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor
antiserum on the one hand and anti-TNF and monoclonal anti-TNF
receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNF stimulation in both cell types. In contrast, a
polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism
and a 50% antagonism to a TNF stimulation in a
dose-dependent fashion, indicating that subtle functional
responses to complex agonist and antagonist stimuli could be measured.
The effects of different proteasome inhibitors to prevent IB
degradation and subsequent NF-B translocation could also be
discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor with
an IC50 of 2 µM, while
clastolactacystin 
-lactone was a complete inhibitor with an
IC50 of 10 µM. The nonselective kinase
inhibitor K252a completely inhibited both IL-1 and TNF
stimulation in both cell types with an IC50 of 0.4 µM. This concentration, determined after a 20-min
stimulation, was shown to be comparable with that obtained for
inhibition of IL-6 production induced by a 100-fold lower IL-1 and
TNF concentration measured after 17 h of stimulation. These
results suggest that the ArrayScanTM technology provides a rapid,
sensitive, quantitative technique for measuring early events in the
signal transduction of NF-B.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
are two
master cytokines that induce an almost identical proinflammatory
response, including the production of chemotactic cytokines, adhesion
molecules, and enzymes such as cyclooxygenase, nitric-oxide synthetase,
and matrix metalloproteinases (1, 2). Many of these effects are a
result of the activation by both IL-1 and TNF of the NF-B
transcription factor pathway, which is associated with the activation
of many cellular defense genes (3, 4). Composed of p65 (RelA) and p50
proteins, NF-B is normally present in the cytoplasm in an inactive
state in a complex with members of the IB inhibitor protein family,
chiefly the 37-kDa IB form. In this complexed form, a nuclear
localization sequence found on NF-B is masked by the IB,
preventing nuclear translocation of NF-B, DNA binding, and
subsequent transcriptional activation (5-12). IL-1 or TNF receptor
activation induces within several minutes the specific phosphorylation
of Ser32 and Ser36 on IB, the destruction
of the phosphorylated IB protein by proteasomes, and the
translocation of NF-B to the nucleus (13-17). Recent reports have
identified an IL-1- and TNF-activated Ser/Thr kinase cascade
containing at least four kinases that serially phosphorylate each other
prior to the phosphorylation of IB (reviewed in Refs. 18 and 19).
The inhibition of proteasome activity by specific inhibitors as LLL-H
(MG132) prevented both the destruction of IB as well as the
subsequent activation of NF-B as measured by the production of
NF-B-dependent proteins such as leukocyte adhesion
proteins (20, 21).
B
activation are difficult because of the rapidity and complexity of the
protein interactions. To show activation of NF-B, electrophoretic mobility shift assays are typically performed to look at the specific binding of activated NF-B to DNA (5, 7, 22), but this technique
requires relatively large numbers of cells and is laboriously quantitative, and the assay is not performed in intact cells. In
contrast, the use of gene reporter constructs in transfected cells
measures a response occurring hours after cell activation, and the
resulting gene transcription is influenced by other transcription factors that act cooperatively to activate individual genes (12, 23).
Because protein translocation from the cytoplasm to nucleus can be
readily visualized by immunocytolocalization (see e.g. Refs.
9, 10, 24, and 25), a computerized cytometric fluorescence system,
termed the ArrayScanTM, has been developed to analyze translocation of
cytoplasmic proteins in cells grown in 96-well plates (26). We have
used this ArrayScanTM system to quantitate the rate and extent of
NF-B translocation following stimulation of varying IL-1 and TNF
concentrations. We show that in human chondrocytes and HeLa cells
complete translocation of NF-B occurs within 10-20 min, with a
half-time several minutes following that of IB destruction. Furthermore, the effects of receptor agonists and antagonists and
kinase and proteasome inhibitors can be differentially quantitated.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B translocation (data not shown). The compounds used together
with their source included K252a (Alexis Biochemicals, San Diego, CA),
carbobenzoxy-Leu-Leu-Leu-aldehyde (Peptide Institute, Osaka, Japan),
and clastolactacystin -lactone (Affiniti Research Products, Mamhead,
Exeter, United Kingdom). Protein antagonists were added in Dulbecco's
modified Eagle's medium and included IL-1 receptor antagonist (IL-1RA;
prepared at Merck), neutralizing anti-TNF and monoclonal anti-TNFR1
and anti-TNFR2 antibodies (R & D Systems, Minneapolis, MN). Polyclonal IL-1R1, anti-TNFR1, and anti-TNFR2 antibodies were prepared at Covance
(Denver, PA) from soluble IL-1R1, TNFR1, and TNFR2 (R & D Systems). All
samples were performed at least in duplicate. The cells were
preincubated for 20 min at 37 °C, and then 10 µl of stimulator
(typically 10 ng/ml (final concentration) IL-1 or TNF; R&D
Systems) was added for each well. The cells were repipetted twice for
mixing and then incubated for typically 20 min more at 37 °C.
Experiments were ended by washing the plates twice in ice-cold
phosphate-buffered saline followed by fixation.
B antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) diluted 1:2000 in 0.1 M phosphate buffer, pH 7.8, with 0.1% bovine serum albumin (fraction V; Sigma). The plates were washed three times in Tris wash
buffer and incubated 30 min, room temperature, with 100 µl of a 10 µg/ml solution in water of biotinylated antirabbit IgG (Vector
Laboratories, Burlingame, CA). The plates were washed three times in
Tris wash buffer and incubated 30 min, room temperature, with 100 µl
of a 2.5 µg/ml solution of Texas Red avidin (Vector) in the
phosphate/bovine serum albumin buffer. The cells were washed three
times in Tris wash buffer and stored in 100 µl of 0.1 M Tris. Two hours prior to analysis 100 µl of a 1 µg/ml solution of
Hoechst 33342 (Molecular Probes, Inc., Eugene, OR) in
phosphate-buffered saline was added to each well at room temperature,
and the wells were scanned in the ArrayScanTM instrument.
B antibody (Santa Cruz Biotechnology)
with ECL visualization as described earlier (28). Quantitative
densitometry was performed using PDI Quantity One software (Bio-Rad) as
previously reported (29).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
B Localization in Cytoplasmic
and Nuclear Cellular Compartments--
Comparable with previous
reports (10, 24), immunofluorescence staining of p65 in unstimulated
HeLa cells and human chondrocytes shows largely a cytoplasmic location
with significantly less nuclear fluorescence (Fig.
1, A and D,
respectively). Following a 20-min stimulation with either IL-1 (Fig. 1,
B and E) or TNF (Fig. 1, C and
F), large amounts of the p65 are found in nuclei, although significant amounts of p65 still remain in the cytoplasm. To quantitate this shift in p65 immunofluorescence, a technology needed to be developed to measure fluorescence separately in both the cytoplasm as
well as the nucleus and to be able to do so in large numbers of cells
in multiple wells. This was achieved with the development of the
ArrayScanTM cytometer as is outlined in Fig.
2. On an individual cell basis, the cells
were stained not only with anti-p65 antibodies to identify NF-B but
also with Hoechst 33342 to identify the nucleus of all of the cells in
the well. The ArrayScanTM cytometer focused on the stained nucleus and
determined the nuclear boundary by the application of a dynamic
thresholding method that identifies a change in fluorescence above the
background (Fig. 2, Step 2). To delineate a
representative nuclear region relatively free of contaminating cytosol,
the nuclear boundary to be analyzed was eroded by 2 pixels (Fig. 2,
Step 3). The resultant nuclear ring is shown in
Fig. 3, where in unstimulated and
IL-1-stimulated HeLa cells (Fig. 3, A and C,
respectively) it is superimposed on the p65 immunofluorescence image
(Fig. 3, B and D). The cytoplasm was identified
by the area lying between a 2-pixel-wide annular ring drawn around the
nuclear boundary and the nuclear boundary itself (Fig. 2,
Step 3). In images of unstimulated (Fig.
4, A-D) and IL-1-stimulated
HeLa cells (Fig. 4, E-H), the cytoplasmic boundary is
denoted by the two red rings.
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Fig. 1.
IL-1- and TNF -induced NF-B nuclear
translocation. Representative fields of p65 fluorescence are shown
in HeLa cells (A-C) and chondrocytes (D-F) that
were unstimulated (A and D), IL-1-stimulated
(B and E), or TNF-stimulated (C and
F). Stimulation was for 20 min with 10 ng/ml cytokine as
described under "Experimental Procedures." Images are
those obtained with the ArrayScanTM cytometer before image analysis
defining nucleus and cytoplasm. The magnification as printed is × 80.
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Fig. 2.
Diagram of the steps taken for quantification
of cytoplasm to nuclear translocation for a given protein by the
ArrayScanTM cytometer. Hoechst 33342 staining defines
the nucleus and enables focusing on the cells within the field
(Step 1). Each cell completely within the field
of view is identified by number, and the nuclear boundary is drawn
(Step 2). To reduce cytoplasmic contamination
within the nuclear area, a nuclear ring 2 pixels inner to the nuclear
boundary is drawn to define the nuclear area used for quantitation. The
outermost cytoplasmic boundary is drawn 2 pixels beyond the nuclear
boundary, and the cytoplasmic area used for quantitation is that which
lies between nuclear boundary and this outer cytoplasmic annular ring.
In both the nuclear and cytoplasmic areas, the intensity of
fluorescence of a given protein is averaged throughout the measured
area. The typical results for a cytoplasmic protein stained in
green are shown at the bottom. Prior to
stimulation, the cytoplasm is intensely green while the
nucleus without the protein is white as is seen from the
side view (viewing from the top shows some nuclear staining because of
cytoplasmic overlap above and below the nucleus). Hence, the nuclear
minus cytoplasmic (Nuc-Cyt Difference) fluorescence is low.
In the stimulated cell, the amount of cytoplasmic fluorescence is
decreased, while the nuclear fluorescence markedly increases, producing
a much darker green in the nucleus due to the
concentration of the cytoplasmic protein. Hence the nuclear minus
cytoplasmic fluorescence is high.
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Fig. 3.
Nuclear identification by Hoechst 33342 staining. Fields of unstimulated (A and B)
and IL-1 -stimulated (C and D) HeLa cells were
visualized by either Hoechst 33342 staining (A and
C) or anti-p65 staining (B and D).
Each cell is shown with an eroded nuclear ring (2 pixels eroded from
the nuclear boundary; see Fig. 2) superimposed upon the nuclear or p65
staining.
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Fig. 4.
Unstimulated (A-D) and
IL-1 -stimulated (E-H) HeLa cells from
ArrayScanTM analysis are shown with the eroded nuclear ring
(blue) together with the cytoplasmic and original nuclear
boundaries (red) used to denote the cytoplasmic region for
analysis.
B (Fig.
5B). Because of the low signal/noise (stimulated
versus unstimulated cell values) observed in both the
nuclear (Fig. 5A) and cytoplasmic regions (Fig.
5B) following stimulation, the individual cellular mean cytoplasmic staining was subtracted from its corresponding nuclear staining. This nuclear/cytoplasmic difference yielded a much more sensitive signal-to-noise ratio (Fig. 5C). Consequently the
nuclear/cytoplasmic difference was typically used as a measure of
NF-B translocation.
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Fig. 5.
Changes in intracellular p65 fluorescence
following IL-1 stimulation of HeLa cells. A, separately
eight unstimulated cells and eight cells stimulated for 20 min with
IL-1 were analyzed to determine the mean amount of p65 fluorescence
in the nucleus. B, the same cells as in A were
analyzed for the amount of mean p65 fluorescence found in the
cytoplasmic area. C, for each of the 16 cells in
A and B, the nuclear minus cytoplasmic (Nuc
Cyt) difference of their pixel-averaged values was determined and
plotted. For each group of cells in A-C, the mean value is
printed above the group.
B Translocation--
To determine how long
the assays needed to be run to achieve maximal NF-B translocation,
HeLa and chondrocyte cells were incubated for up to 45 min following
stimulation by a 25-ng/ml dose of IL-1 or TNF. As is shown in Fig.
6, the HeLa cells responded slightly
faster than the chondrocytes with a time for 50% maximal response
being reached by 7-8 min for TNF and IL-1 stimulation, whereas
the chondrocytes were slightly slower with 50% maximal responses at
12-13 min. For both cells, maximal response was achieved by 20 min.
Hence, for the translocation assays, a 20-min incubation was typically
used for both cell types.
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Fig. 6.
Time course of NF- B nuclear
translocation. Plates of HeLa (open symbols)
and chondrocyte cells (closed symbols) were
stimulated with 25 ng/ml IL-1 (circles) or TNF
(triangles) for the indicated times at 37 °C. The cells
were fixed and stained with anti-p65 antibody and Hoechst 33342 and
analyzed in the ArrayScanTM instrument as described under
"Experimental Procedures." Nuc Cyt, nuclear minus
cytoplasmic.
B translocation follows IB phosphorylation and
degradation, the corresponding effects on IB in the cell types were also studied using IL-1 as a stimulus. As is shown in Fig. 7, IB was almost totally destroyed
by 10 min after stimulation. The time for 50% maximal response was
about 4 min for the HeLa cells and about 7.5 min for the chondrocytes,
indicating that IB proteolysis preceded NF-B nuclear
translocation by 4-5 min.
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Fig. 7.
I B degradation following IL-1
stimulation of chondrocytes and HeLa cells. Cells were stimulated
at 37 °C with 25 ng/ml of IL-1 or TNF for the indicated times
and lysed in sample buffer for SDS-PAGE for determination of IB
by immunoblot (bottom). For each cell type, the immunoblot
density at each time was scanned and plotted as the absorption relative
to that of the unstimulated cells (top). The cells were
grown in 35-mm dishes for IB analysis as described under
"Experimental Procedures."
Dose Response--
Titration of the concentration
of IL-1 and TNF used for cell stimulation indicated that maximal
stimulation of each cytokine occurred at about 10 ng/ml in each cell
type (Fig. 8). The dose response for IL-1
was steeper than that of TNF in both cell types; for IL-
stimulation the ED50 for both cell types was about 1-2 µg/ml, whereas for TNF stimulation the ED50 was about
0.03-0.05 µg/ml.
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Fig. 8.
Dose response of IL-1 - and TNF-induced
NF-B nuclear translocation. The indicated concentrations of
IL-1 (top panel) or TNF (bottom
panel) were added to cultures of chondrocytes (
) or HeLa
cells (
) incubated for 20 min 37 °C and then assayed by the
ArrayScanTM. Nuc Cyt, nuclear minus cytoplasmic.
-stimulated chondrocytes were analyzed by two-way analysis
of variance. In these experiments, the mean difference between nuclear
and cytoplasmic p65 staining in stimulated cells was about 55 units
more than in unstimulated cells. The number of cells needed for
analysis to be assured that a change of 90% had occurred
(i.e. a nuclear/cytoplasmic difference of 49.5 (0.9 × 55)) was 56 at the 99% confidence level (i.e. a 1% false
positive, type I error; see Table I).
This was true regardless of whether the change was due to the addition
of varying amounts of a cytokine stimulator or to varying amounts of an
inhibitor added to a 10-ng/ml cytokine stimulation. For 95% confidence
that the 90% inhibition was achieved, a total of only 37 cells was
required to be analyzed. For statistically significant smaller changes
in the nuclear/cytoplasmic difference, correspondingly larger numbers
of cells needed to be analyzed to achieve comparable confidence (Table
I). If only the change in nuclear p65 staining were analyzed, rather
than the nuclear/cytoplasmic difference, then about 9% more cells were required to be analyzed to give the same statistical significance (data
not shown). Based upon these data, a cell number of 75 was typically
analyzed in the experiments described in this paper.
Number of cells needed in the nuclear translocation assay to detect
differences at specified levels of confidence
were analyzed to
determine the number of cells required for statistical significance at
the 99 and 95% confidence levels for the indicated percentage change
of a 55-unit nuclear/cytoplasmic difference. The false negative (Type
II) error was assumed to be 20%.
in the presence or absence of an IL-1RA
antagonist were analyzed by the ArrayScanTM system. Reproducibility of
this assay for screening purposes was assessed by calculating the
coefficient of variation between IC50 (with IL-1RA) or
EC50 (without IL-1RA) values generated from 11 point curves
in each row (n = 8) of a 96-well microtiter plate. In
this way, row to row, plate to plate, and day to day variability was
measured and did not exceed 20, 30, and 22%, respectively (Table
II). The reproducibility of the detection
method was evaluated by repeatedly scanning the same plate five
times, yielding after analysis a coefficient of variation of 6.8%
(Table II).
Sources of variability and their relative magnitude in the ArrayScanTM
nuclear translocation assay
.
B
Translocation--
IL-1RA (30) inhibited the NF-B translocation
with an IC50 of 20-60 nM, a concentration
about 100-fold higher than the stimulatory IL-1 concentration (Fig.
9, top), but it had no effect
on the activation of NF-B translocation induced by TNF (not
shown). This IC50 concentration of IL-1RA determined by the
ArrayScanTM method was comparable with that shown earlier to be
required to inhibit IL-1-driven cellular responses (31). For complete
IL-1 antagonism, 1000-fold higher IL-1RA concentrations were required to prevent NF-B translocation (Fig. 9, top), just as were
seen in other bioassays (32). In contrast, a neutralizing anti-TNF antibody blocked TNF stimulation of NF-B translocation (Fig. 9,
bottom) but had no effect on IL-1 stimulation (not
shown). The blockage of IL-1 stimulation of NF-B translocation by
IL-1RA occurred at the same concentration as that necessary to block IB degradation. In an experiment in which IB degradation
was quantitated in a similar plate in which NF-B translocation was measured, both assays yielded the same 24 nM IL-1RA
concentration for 50% inhibition (Fig.
10). This gives further evidence of the close correlation of NF-B translocation to the degradation of IB.
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Fig. 9.
Inhibition of NF- B translocation by IL-1RA
and anti-TNF antibody in chondrocytes () and HeLa cells
(). Top, cells were stimulated with 10 ng/ml IL-1
for 20 min at 37 °C in the presence of the indicated concentration
of IL-1RA. Bottom, cells were stimulated for 20 min at
37 °C with 10 ng/ml TNF that had been preincubated 30 min at room
temperature with the indicated concentration of a neutralizing
anti-TNF antibody. Nuc Cyt, nuclear minus
cytoplasmic.
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Fig. 10.
Comparison of the extent of inhibition of
IL-1-induced I B degradation and NF-B translocation by
IL-1RA. Parallel 96-well plates of chondrocytes were stimulated
for 20 min 37 °C with 10 ng/ml IL-1 in the presence of the
indicated concentration of IL-1RA in columns 1-12 of the plate (see
"Concentrations for the Immunoblots"). All eight rows were
replicates. For the IB blots, all eight replicates of each
concentration from one plate were combined with SDS-PAGE sample buffer
to concentrate the cells to perform the indicated SDS-PAGE and
immunoblot. The scanned intensity of the IB blot was plotted
(, top panel) and compared with the NF-B
translocation obtained from ArrayScanTM analysis of the other plate
(
, top panel). Nuc Cyt, nuclear
minus cytoplasmic.
B translocation could be prevented by serial dilutions of an
anti-IL-1R1 antiserum but not by a preimmune bleed (Fig.
11, A and C,
respectively). The antiserum on its own had no agonist activity,
indicating that it was a strict antagonist. Similarly, a neutralizing
monoclonal antibody against the TNFR1 blocked TNF-induced NF-B
translocation, but it had no agonism of its own, and a monoclonal
anti-TNFR2 antibody was ineffective (Fig. 11, B and
D). This indicated that all of the functional TNF receptors
in these cell types were TNFR1. In contrast, when a polyclonal
anti-TNFR1 antibody was added with chondrocytes stimulated with TNF,
it showed both partial antagonism and partial agonism (Fig.
11E). This antiserum in the absence of TNF gave roughly a
maximal 50% stimulation of the NF-B translocation, while in the
presence of the TNF stimulus, the same 50% of NF-B translocation was seen. Titration of the antiserum down resulted in either an increasing loss of NF-B translocation (without TNF stimulation) or a comparable increase in NF-B translocation (samples with TNF
present; Fig. 11E). The IC50 values were the
same for stimulation and inhibition, suggesting that the same
antibodies that could block a TNF-induced activation could on their
own activate the cells.
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Fig. 11.
Inhibition of NF- B nuclear translocation
by IL-1R1- and TNFR1/2-neutralizing antibodies. Chondrocytes
(A) or HeLa cells (C) were preincubated with
various dilutions of a rabbit polyclonal anti-IL-1R1 antiserum
(closed symbols) or preimmune serum
(open symbols) followed by the absence
(circles) or presence (triangles) of IL-1
stimulator. Similarly, chondrocytes (B) or HeLa cells
(D) were preincubated with various dilutions of either
monoclonal anti-TNFR1 (closed symbols) or
anti-TNFR2 (open symbols) antibodies followed by
the absence (circles) or presence (triangles) of
TNF stimulator. E, chondrocytes were preincubated with
polyclonal anti-TNFR1 (, ), a preimmune control (
, 
), or
polyclonal anti-TNFR2 (, 
) antisera followed by the absence (,
, ) or presence (, , ) of TNF. Nuc Cyt,
nuclear minus cytoplasmic.
B
Translocation--
Because proteasome inhibitors prevent IB
degradation (16, 33), the potent proteasome inhibitors LLL-H (20) and
clastolactacystin -lactone (34) were tested for their potency as
NF-B translocation inhibitors in chondrocytes and HeLa cells
stimulated with IL-1 and TNF. As is shown in Fig.
12, clastolactacystin -lactone gave complete inhibition of translocation with an IC50 of about
10 µM. In contrast, LLL-H only inhibited maximally about
of the total translocation, and this occurred with an
IC50 of about 2 µM. The only partial
inhibitory effect of LLL-H on NF-B translocation was also seen
correspondingly on IB degradation; concentrations as high as 25 µM LLL-H only delayed rather than blocked IB
degradation (data not shown; see Ref. 28).
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Fig. 12.
Inhibition of NF- B translocation by
proteasome inhibitors. Chondrocytes (closed
symbols) and HeLa cells (open symbols)
were stimulated with IL-1 (circles) or TNF
(triangles) in the presence of the indicated concentration
of carbobenzoxy-Leu-Leu-Leu-aldehyde (top panel)
or clastolactacystin -lactone (bottom panel)
as described under "Experimental Procedures" followed by
ArrayScanTM analysis. Nuc Cyt, nuclear minus
cytoplasmic.
B (28). The compound K252a (which is more cell-permeable than
K252b) was tested for its effects on NF-B translocation in
chondrocytes and HeLa cells stimulated with IL-1 or TNF and analyzed
by the ArrayScanTM. As is shown in Fig.
13A, the IC50
for both cell types and both stimuli in the ArrayScanTM assay was about 0.4 µM. To compare the inhibition of a compound such as
K252a in the ArrayScanTM assay to its effects in a traditional assay measuring inhibition of an NF-B-up-regulated and -secreted protein, the effects of K252a on the IL-1- and TNF-induced production of IL-6
in MRC-5 cells were determined in an overnight assay. This normal human
fibroblast MRC-5 cell line is highly sensitive to both cytokines,
IB is rapidly phosphorylated and degraded (28), and the MRC-5
cells behaved comparably with chondrocytes when tested in the
ArrayScanTM technique (data not shown), factors making them an
excellent choice for comparison with chondrocytes and HeLa cells. K252a
was found to inhibit the MRC-5 cells at an IC50 of
approximately 0.2-0.3 µM (Fig. 13B), about
the same as the chondrocytes and HeLa cells (Fig. 13A).
View larger version (30K):
[in a new window]
Fig. 13.
Inhibition of NF- B translocation and IL-6
production by the kinase inhibitor K252a. A,
chondrocytes (solid symbols) or HeLa cells
(open symbols) were stimulated with IL-1
(circles) or TNF (triangles) in the presence
of the indicated concentrations of K252a as described under
"Experimental Procedures" followed by ArrayScanTM analysis.
B, effect of K252a on the inhibition of IL-6 production in
MRC-5 cells when stimulated by IL-1 () or TNF (). MRC-5
cells were stimulated for 17 h with 100 pg/ml cytokine, and the
amount of IL-6 production was determined by enzyme-linked immunosorbent
assay kits (Genzyme, Cambridge, MA). Because of the longer time of the
assay, a 100-fold lower concentration of stimulatory cytokines was
used. Nuc Cyt, nuclear minus cytoplasmic.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
signal transduction in intact cells
by measuring translocation of NF-B from cytoplasm to the nucleus.
Cellular analysis of NF-B translocation is performed within 20 min
following the synchronized stimulation of cells with IL-1 and TNF.
This rapid response focuses on just the process leading up to the
appearance of NF-B in the nucleus prior to any nuclear binding and
expression of its transcription activity. The assay thus reduces the
number of postreceptor, intracellular signaling steps affecting the
measurement to those of the kinase and adaptor protein cascade
producing IB phosphorylation, the IB destruction by
proteasomes, and the unknown factors enabling nuclear entry of NF-B.
As is shown in this report, ArrayScanTM analysis of NF-B
translocation is highly quantitative, sensitive, and reproducible. The
low coefficient of variation (6.8%) for repeatedly scanning the same
wells (but different fields of cells) indicates the ability of the
system to extract statistically similar data from a number of fields in
the same well. The analysis of variance indicated that this nuclear
translocation technique was robust and produced only a minimal
variation between rows and between plates regardless of whether they
were analyzed on the same or successive days. Assay, plate, and day
variability (Table II) were similar to those obtained for other
cell-based assay formats currently used in compound screening.
ArrayScanTM analysis is also rapid, taking less than 1 h to scan
75 cells/well in a 96-well plate. Furthermore, determination of protein
distribution can be made on native, untransfected cells using fixation
and staining such as that described here, or dynamic analysis can be
performed in living cells in which proteins of interest are labeled
with a fluorescent probe such as green fluorescent protein attached to
receptors like the glucocorticoid receptor (26, 35) or to other
transcription factors such as NF-AT (25).
B translocation was chiefly identified indirectly by
electrophoretic mobility shift assays where measurement could only
occur following subsequent NF-B binding to DNA, and quantitation
could only be made following cellular lysis, fractionation, and
separation by gel electrophoresis (5, 7, 22). Such a technique is
tedious, multistep, and subject to artifacts of protein partitioning
and separation (36). Analysis with the present ArrayScanTM technique
preserves the spatial distribution of proteins within the cell without
disruption. Electrophoretic mobility shift assay measurement is also
subject to the influences on other components of the transcription
apparatus that are necessary for NF-B binding to the DNA (37).
Hence, results of electrophoretic mobility shift assay analysis may
reflect the influences of a number of signaling pathways rather than
that of NF-B alone. On the other hand, with the ArrayScanTM
measurement of NF-B at an earlier stage in signaling, the comparison
of results by ArrayScanTM analysis with that of measurement of
transcriptional activity of NF-B may enable a clearer understanding
of those events activating NF-B in the nucleus such as
phosphorylation (38).
-stimulated nuclear NF-B
translocation begins within 2-4 min following the phosphorylation of
IB, which is initiated 1-2 min after stimulation (see also Ref.
28). The half-time for nuclear NF-B translocation occurs at about
7-12 min, depending on the cell type, and occurs about 4-5 min
following IB degradation. This time course is comparable for both
IL-1 and TNF activation despite the differences in the nature of the
receptors and the different adaptor proteins involved (see Ref. 18).
The NF-B translocation occurs gradually and uniformly in the entire
cell population during the course of the cytokine stimulation. Only a
small amount of the entire pool of NF-B in the cytoplasm
translocates despite the proteolysis of essentially all of the
associated IB; the cytoplasmic fluorescence decreases by only
20% following stimulation (Fig. 5). This observation is comparable
with earlier observations in which only 10-20% of the cytoplasmic
NF-B was observed to translocate (11). One might consider an
alternative scenario producing the same average nuclear/cytoplasmic
difference in which say only 50% of the cells responded but where the
amount of the NF-B translocation in those cells was twice as much.
If this were the case, however, then the S.D. in the
nuclear/cytoplasmic difference values would be increased over the
course of the translocation. This increase is not seen; the S.D.
between cells is constant during the entire translocation (data not
shown).
produce a maximal response when incubated at about
10 ng/ml (0.6 nM), but they show substantial differences in
the concentrations for half-maximal response; whereas IL-1 titrates
over about 1.5 logs with an ED50 of about 2 ng/ml, the TNF titrates over 3 logs with an ED50 of about 0.15 ng/ml (Fig. 8). These titrations in NF-B nuclear translocation are
comparable with other cellular responses induced by both IL-1 (see Fig.
13) and TNF (see Ref. 39) and are similar to that observed for cytokine receptor binding for both IL-1 (40,
41)2 and TNF (42).
Precisely why TNF is effective in receptor ligation and cell
stimulation over such an extended concentration range is unknown but is
perhaps a function of its trimeric nature (43-45). The antagonism of
the IL-1 response by IL-1RA as measured in the ArrayScanTM faithfully
replicates its antagonism of IL-1 receptor binding, where in both cases
roughly a 100-fold excess of IL-1RA is needed for antagonism. The
agonism induced by the aggregation of the TNFR1 molecules by the
polyclonal antiTNFR1 mimics the TNF response as reported earlier
(39), but quantitation by the ArrayScanTM shows that this agonism is
only partial and is mixed with partial antagonism.
B
translocation at two measurable stages following receptor activation: at the level of the kinase signaling cascade and at the level of
proteosome involvement in IB destruction and liberation of NF-B. The nonspecific kinase inhibitor K252a (46) has been shown to
inhibit NF-B activation (47), and the closely related compound K252b
inhibits both the IL-1 receptor association kinase and the
phosphorylation of IB (28). As is shown in Fig. 13, K252a
inhibits TNF signaling equally well as IL-1 signaling, indicating
that it must inhibit other kinases in the NF-B activation cascade
that are common to both stimulators. As expected from the necessary
role for proteasomes in the degradation of phospho-IB prior to
NF-B nuclear translocation, proteasome inhibitors such as LLL-H (20)
and lactacystin (48) equally prevent both IL-1 and TNF-induced cell
activation (Fig. 12). Because of the ease of quantitation of the
NF-B translocation in the ArrayScanTM, however, subtle differences
between the two proteasome inhibitors can be seen such that LLL-H gives
only partial inhibition of NF-B translocation, while the lactacystin
analog clastolactacystin -lactone gives complete inhibition in both
HeLa and chondrocyte cells. This suggests that there may be at least
two subsets of proteasomes operating in these cells with only one of
them inhibited by LLL-H. Such an interpretation is supported by recent
evidence that cells resistant to LLL-based proteasome inhibitors,
lacking enzymatic activity associated with this class of proteasomes, can still process substrates such as peptides for assembly of class I
histocompatibility antigens. These resistant cells contain a different
high molecular weight proteasome-like complex with a different
substrate specificity (49).
FOOTNOTES
To whom all correspondence should be addressed: Dept. of
Immunology and Inflammation, Merck Research Laboratories, P.O. Box 2000, R80N-A32, Rahway, NJ 07065. Tel.: 732-594-6838; Fax:
732-594-3111; E-mail: douglas_miller{at}merck.com.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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