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Outside-to-inside signaling through transmembrane tumor necrosis factor reverses pathologic interleukin-1 production and deficient apoptosis of rheumatoid arthritis monocytes.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 9, September 2009, pp 2612–2621
DOI 10.1002/art.24778
© 2009, American College of Rheumatology
Outside-to-Inside Signaling Through Transmembrane Tumor
Necrosis Factor Reverses Pathologic Interleukin-1␤ Production
and Deficient Apoptosis of Rheumatoid Arthritis Monocytes
Undine Meusch, Manuela Rossol, Christoph Baerwald, Sunna Hauschildt, and Ulf Wagner
ing inhibited the constitutive NF-␬B activation in RA
monocytes, suppressed IL-1␤ secretion, and normalized
spontaneous in vitro apoptosis. This normalization was
reversible by the addition of exogenous IL-1␤.
Conclusion. This study demonstrates that
outside-to-inside signaling through transmembrane
TNF after ligation by infliximab inhibits constitutive
NF-␬B activation and suppresses spontaneous IL-1␤
production by monocytes from patients with RA. Besides the induction of monocyte apoptosis, this inhibition could also contribute to the therapeutic effects
observed during treatment with TNF␣ inhibitors.
Objective. Monocytes are a major source of proinflammatory cytokines in rheumatoid arthritis (RA), and
inhibitors of monocytic cytokines are highly efficient
agents for treatment of the disease. The aim of this
study was to analyze the effects of a therapeutic anti–
tumor necrosis factor ␣ (anti-TNF␣) antibody on monocytes from patients with RA and healthy control subjects.
Methods. Peripheral blood monocytes from patients with RA and healthy control subjects were incubated in the presence of anti-TNF␣ antibody or IgG.
Annexin V staining, caspase activation, poly(ADP-ribose)
polymerase cleavage, and DNA staining with propidium
iodide were used to analyze apoptosis. The signaling events
elicited in monocytes by infliximab were analyzed by
Western blotting and electromobility shift assay.
Results. Peripheral blood monocytes from patients with RA were characterized by increased expression of transmembrane TNF␣, spontaneous in vitro
production of interleukin-1␤ (IL-1␤), and a decreased
rate of spontaneous ex vivo apoptosis. Incubation with
infliximab induced significantly increased apoptosis in
monocytes from patients with RA but not in monocytes
from healthy control subjects. This apoptosis was triggered by reverse signaling of transmembrane TNF after
ligation by infliximab and was independent of caspase
activation. Instead, transmembrane TNF reverse signal-
The cytokine tumor necrosis factor ␣ (TNF␣)
plays a pivotal role in the pathogenesis of rheumatoid
arthritis (RA). Following de novo synthesis, the cytokine
is integrated in the cell membrane as a type II transmembrane protein arranged in stable homotrimers and
is released in its soluble form exclusively after proteolytic cleavage from this membrane-integrated form by
the metalloproteinase TNF␣-converting enzyme. Transmembrane TNF␣, besides being a precursor of the
soluble cytokine, has also been shown to exert an
important immunologic function on its own.
TNF-binding compounds are now widely used
in clinical practice, and several cellular mechanisms for
their beneficial effects have been described, including
complement-dependent cytotoxicity, antibody-dependent
cell-mediated cytotoxicity, and outside-to-inside (reverse)
signal transduction through transmembrane TNF (1). Our
group previously showed that the cross-talk between transmembrane TNF and its 2 receptors during direct cell
contact of monocytes with preactivated T cells results in
monocyte activation (2). Reverse signaling of transmembrane TNF on the monocyte cell surface was observed to
partially mediate this effect. Other groups of investigators
have shown that reverse signaling induces the expression of
E-selectin in activated CD4⫹ T cells (3), mediates resis-
Supported by grants from the German Ministry for Education
and Science (Interdisziplinäres Zentrum für Klinische Forschung
Leipzig, Teilprojekt A 21).
Undine Meusch, Manuela Rossol, PhD, Christoph Baerwald,
MD, Sunna Hauschildt, PhD, Ulf Wagner, MD: University of Leipzig,
Leipzig, Germany.
Ms Meusch and Dr. Rossol contributed equally to this work.
Address correspondence and reprint requests to Ulf Wagner,
MD, Division of Rheumatology, Department of Internal Medicine II,
University of Leipzig, Liebigstrasse 20, 04103 Leipzig, Germany.
E-mail: ulf.wagner@medizin.uni-leipzig.de.
Submitted for publication December 22, 2008; accepted in
revised form June 1, 2009.
2612
EFFECTS OF ANTI-TNF␣ ANTIBODY ON MONOCYTES FROM PATIENTS WITH RA
tance to lipopolysaccharide (LPS) in monocytes (4), and
influences cytokine production (5,6). However, in other in
vitro settings, the main cellular effect of reverse signaling
following ligation of transmembrane TNF in monocytes
was the induction of apoptosis (6–9). The reported signal
transduction pathways include activation of MAPK p38
(9), autocrine production of transforming growth factor ␤1
(9), activation of caspases 8, 9, and 3 (8,9), and cleavage of
poly(ADP-ribose) polymerase 1 (PARP-1) (9). In view of
those conflicting results, the goal of our study was to
investigate the cellular response to and the proapoptotic
effects of transmembrane TNF reverse signaling in monocytes from healthy individuals and patients with RA, as well
as the underlying signal transduction events.
PATIENTS AND METHODS
Patients and control subjects. Peripheral blood samples obtained from 48 patients with a diagnosis of RA according to the American College of Rheumatology (formerly, the
American Rheumatism Association) 1987 revised criteria for
the classification of RA (10) were analyzed. The study design
was approved by the ethics committee of the University of
Leipzig, and informed consent was obtained from each patient
before study enrollment. Eighty-eight percent of the patients
had IgM rheumatoid factor–seropositive disease, and 81% of
the patients had anti–cyclic citrullinated peptide antibodies.
The median age of the patients was 66 years (range 22–91
years), the median age at disease onset was 52 years (range
18–69 years), and the median disease duration was 14 years
(range 1–38 years). The patients did not receive any TNF␣inhibiting therapy before study enrollment. Current treatment
regimens included conventional disease-modifying antirheumatic drugs, given either as monotherapy or in combination, as
well as low-dose prednisolone in 80% of the patients.
Fifty age-matched control subjects with no history of
inflammatory arthritis were recruited from among healthy
blood donors, with ethics committee approval. The median age
of the control subjects was 34 years (range 23–65 years).
Monocyte isolation, cell culture, and stimulation of
human monocytes. Monocytes were isolated as described
previously (11). Monocytes (2 ⫻ 105/200 ␮l) were incubated in
RPMI 1640 supplemented with 5% human AB serum (heat
inactivated) and either 100 ␮g/ml IgG (Intraglobin CP; Biotest,
Dreieich, Germany) or infliximab (Centocor, Leiden, The
Netherlands) for 16 hours. In some experiments, the soluble
TNFR2:Ig construct etanercept (Wyeth-Pharma, Münster,
Germany) was used instead of infliximab. For the inhibition of
casein kinase 1 (CK-1), cells were preincubated with the CK-1
inhibitor D4476 (150 ␮M; Calbiochem, La Jolla, CA) for 15
minutes. For global inhibition of caspases, the pan-specific
inhibitor Z-VAD-FMK (10 ␮M; Calbiochem) was used. To
inhibit caspase 3, the cells were incubated with the inhibitor
Z-DEVD-FMK (10 ␮M; Calbiochem).
Detection of transmembrane TNF by flow cytometry.
Monocytes (2 ⫻ 105/50 ␮l) were incubated with 1 mg/ml IgG
(Intraglobin CP; Biotest) in phosphate buffered saline (PBS)
2613
supplemented with 2% fetal calf serum (FCS) and 0.1%
Na-azide for 30 minutes at 4°C to block Fc receptor binding.
Subsequently, phycoerythrin-labeled anti-TNF antibody (clone
6402.31) or IgG1 isotype control (R&D Systems, Minneapolis,
MN) was added, and cells were incubated for an additional 30
minutes at 4°C. After the cells were washed with PBS supplemented with 2% FCS and 0.1% Na-azide, they were fixed with
1% formaldehyde. Transmembrane TNF expression was analyzed by flow cytometry (FACSCalibur; BD Biosciences, San
Jose, CA), and analysis was performed using CellQuest software (BD Biosciences).
Determination of apoptosis. Apoptotic and necrotic
cells were stained with 10 ␮l fluorescein isothiocyanate–
labeled annexin V (Southern Biotechnology, Birmingham, AL)
and 50 ␮g/ml propidium iodide (PI), respectively. Flow cytometry was performed using the FACSCalibur system, and the
results were analyzed using CellQuest software.
Measurement of DNA degradation. Monocytes (1 ⫻
106/ml) were washed with PBS supplemented with 0.05%
Na-azide and incubated with 4% paraformaldehyde for 15
minutes at 4°C. Cells were washed again with PBS supplemented with 0.05% Na-azide and subsequently incubated in
0.1% saponin and 62.5 ␮g/ml PI for 20 minutes at 4°C. Flow
cytometry was performed using the FACSCalibur system, and
results were analyzed using CellQuest software.
Gel electrophoresis and Western blotting. Gel electrophoresis and Western blotting were performed as described
previously (2). Rabbit monoclonal antibodies for the detection
of caspase 3, Bcl-xL, and phosphorylated I␬B␣, mouse monoclonal antibodies for the detection of cleaved PARP and
caspase 8, and polyclonal rabbit antibodies for the detection of
caspase 9, caspase 1, and interleukin-1␤ (IL-1␤) were purchased
from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal
anti-human I␬B␣ antibody and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Preparation of nuclear extracts. Nuclear extracts were
prepared from 6 ⫻ 106 monocytes after incubation with IgG or
infliximab for 16 hours. Cells were washed with PBS supplemented with 1 mM EDTA, resuspended in lysis buffer containing 10 mM Tris (pH 7.8), 5 mM MgCl2, 10 mM KCl, 1 mM
EGTA (pH 7.0), 10% sucrose, 1 mM dithiothreitol, 10 mM
␤-glycerophosphate (Sigma-Aldrich, St. Louis, MO), 1 mM
phenylmethylsulfonyl fluoride (PMSF), and Complete mini
protease inhibitors (Roche Diagnostics, Mannheim, Germany), placed on ice for 15 minutes, and then vortexed for 10
seconds after the addition of 25 ␮l of 10% Nonidet P40. After
a 5-minute centrifugation (1,700g at 4°C), the pelleted nuclei
were gently washed with 200 ␮l lysis buffer without Nonidet
P40 and pelleted (1,700g for 1 minute at 4°C). Pelleted nuclei
were resuspended in 30 ␮l extraction buffer containing 20 mM
Tris (pH 7.8), 5 mM MgCl2, 320 mM KCl, 0.2 mM EGTA, 25%
glycerol, 0.1 mM dithiothreitol, 10 mM ␤-glycerophosphate, 1
mM PMSF, and Complete mini protease inhibitors. The
nuclear extracts were maintained on ice for 15 minutes, with
occasional vortexing. Subsequently, nuclear extracts were
cleared (20,800g for 5 minutes at 4°C) and transferred to fresh
tubes. Protein concentrations were determined with a
detergent-compatible protein assay (Bio-Rad, Hercules, CA).
All extracts were stored at –80°C.
Electrophoretic mobility shift assay. Single-stranded
oligonucleotides containing the sequences corresponding to
2614
MEUSCH ET AL
the NF-␬B consensus site (5⬘-AGTTGAGGGGACTTTCCCAGGC-3⬘ and 3⬘-TCAACTCCCCTGAAAGGGTCCG-5⬘
[the binding site sequence is underlined]) were synthesized by
personnel at MWG Biotech (Ebersberg, Germany). To determine whether the observed shifted bands are specific for
NF-␬B, the following mutant oligonucleotide probes were
used: for NF-␬B, 5⬘-AGTTGAGGCGACTTTCCCAGGC-3⬘
and 3⬘-TCAACTCCGCTGAAAGGGTCCG-5⬘ (the binding
site sequence is underlined, and the mutated site is shown in
bold). Single-stranded oligonucleotide probes were endlabeled with [␥-32P]-ATP using T4 polynucleotide kinase and
separated from the unincorporated label using the QIAquick
Nucleotide Removal Kit (Qiagen, Hilden, Germany). The
purified sense and antisense oligonucleotide probes were
annealed for 10 minutes at room temperature to a final
concentration of 2 ␮g/ml. The protein–DNA binding reaction
was carried out in a volume of 10 ␮l containing binding buffer
(10 mM Tris [pH 8.0], 150 mM KCl, 0.5 mM EDTA, 0.1%
Triton X-100, 12.5% glycerol, 0.2 mM dithiothreitol), 2 ␮g of
poly(dG-dC), 1 ␮g of poly(dA-dT) (both from Sigma-Aldrich),
2 ng of labeled double-stranded oligonucleotide probe, and 5
␮g of nuclear extract. The binding reaction was allowed to
proceed for 30 minutes at room temperature. Samples were
subjected to electrophoresis in 5% nondenaturating polyacrylamide gel in a 0.5⫻ Tris–borate–EDTA buffer system for 2.5
hours at 4°C at 150V. After electrophoresis, the gel was
transferred to Whatman paper, dried on a gel dryer for 2 hours
at 80°C, and visualized using the FLA-3000 Phosphor Imaging
System (Fujifilm, Duesseldorf, Germany).
Detection of IL-1␤ and IL-1 receptor antagonist (IL1Ra). IL-1␤ and IL-1Ra concentrations were measured by
using an IL-1␤ enzyme-linked immunosorbent assay (ELISA)
from BD Biosciences and an IL-1Ra ELISA from R&D
Systems, according to the manufacturer’s instructions.
Statistical analysis. For statistical analysis, SigmaStat
software (SPSS, Chicago, IL) was used. Prior to all comparisons, a normality test was performed. To assess statistical
significance, Student’s t-test (normal distribution of data) or
the Mann-Whitney rank sum test (unequal distribution of
data) was used. Correlation of 2 parameters was analyzed with
Pearson’s product-moment correlation.
RESULTS
High-level transmembrane TNF␣ expression by
peripheral blood monocytes from patients with RA. Our
group has previously demonstrated that ligation of transmembrane TNF on monocytes triggers reverse signaling
and induction of TNF␣. Such reverse signaling is a
strong stimulus for monocytes during direct cellular
contact with preactivated T cells and can be inhibited by
anti-TNF antibodies (2). To investigate a potential role
for transmembrane TNF in patients with RA, cell surface expression of the molecule was analyzed by flow
cytometry.
Monocytes from healthy donors and patients with
RA expressed transmembrane TNF on the cell surface,
Figure 1. Expression of transmembrane tumor necrosis factor
(tmTNF) on freshly isolated monocytes from healthy donors (HD;
n ⫽ 21) and patients with rheumatoid arthritis (RA; n ⫽ 23), as
determined using flow cytometry. A, Expression on monocytes stained
with phycoerythrin-labeled IgG1 control antibody (open histogram) or
anti-TNF antibody (shaded histogram). B, Dot plot depicting the
median fluorescence intensity of transmembrane TNF surface expression. Bars show the median.
as shown in representative histograms in Figure 1A. A
comparison of the median fluorescence intensity of
monocytes from healthy donors and patients with RA
showed significantly higher expression of transmembrane TNF on monocytes from patients with RA
(P ⫽ 0.012), while monocytes from healthy donors expressed no or only low levels of transmembrane TNF
(Figure 1B).
Spontaneous in vitro production of IL-1␤ increased in monocytes from RA patients and inhibited by
transmembrane TNF reverse signaling. The cytokine
IL-1␤ plays a pivotal role in monocyte biology and has
both proinflammatory functions and an antiapoptotic
effect. Therefore, in vitro IL-1␤ production in culture
supernatants was analyzed by ELISA. After 4 hours of in
vitro culture, monocytes from patients with RA spontaneously produced significant amounts of IL-1␤, whereas
monocytes from healthy donors did not (Figure 2A).
Increased IL-1␤ levels were also observed in sera from
patients with RA, while no IL-1␤ was detected in sera
from healthy donors (Figure 2B). When RA monocytes
were incubated in the presence of infliximab, IL-1␤
concentrations were reduced to levels comparable with
those in monocytes from healthy donors (Figure 2A).
Correspondingly, protein expression of inactive procaspase 1, the IL-1␤–converting enzyme, and of
proIL-1␤ as determined by Western blot analysis was
significantly increased following the addition of infliximab (Figure 2C). In contrast, spontaneous production
of IL-1Ra did not differ between monocytes from
healthy donors and those from patients with RA and
was not influenced by the addition of infliximab
(Figure 2D).
EFFECTS OF ANTI-TNF␣ ANTIBODY ON MONOCYTES FROM PATIENTS WITH RA
2615
optosis during in vitro culture revealed that the percentage of apoptotic cells was significantly decreased in
monocytes from patients with RA compared with control subjects (Figure 3A). To eliminate the influence of
a prolonged in vitro culture period on this result, freshly
separated CD14⫹ peripheral blood monocytes were
analyzed ex vivo for apoptosis. Again, monocytes from
healthy individuals were more frequently annexin V
positive than were monocytes from patients with RA
(mean ⫾ SEM 14.3 ⫾ 1.3% [n ⫽ 6] and 10.6 ⫾ 0.6%
[n ⫽ 7], respectively; P ⫽ 0.019).
Increased in vitro apoptosis of monocytes by
transmembrane TNF reverse signaling. In previous
studies investigating reverse signaling via transmem-
Figure 2. Inhibition of interleukin-1␤ (IL-1␤) production in monocytes from patients with RA by transmembrane TNF reverse signaling.
A and D, Production of IL-1␤ (A) and IL-1 receptor antagonist
(IL-1Ra) (D) by monocytes from patients with RA (n ⫽ 7) and healthy
donors (n ⫽ 4). Cells were incubated for 4 hours in the presence of
infliximab (inflix.) or IgG. Bars show the mean and SEM. B, IL-1␤
concentration in sera from healthy donors (n ⫽ 31) and patients with
RA (n ⫽ 32), as analyzed by enzyme-linked immunosorbent assay.
Bars show the median. C, Expression of procaspase 1 and proIL-1␤ in
monocytes from patients with RA, as analyzed by Western blotting
after 4 hours of incubation with infliximab or IgG. Results are
representative of 3 experiments. See Figure 1 for other definitions.
In order to assess the net effect of infliximabinduced transmembrane TNF reverse signaling on the
IL-1␤/IL-1Ra system, the ratio of the IL-1␤ to IL-1Ra
concentration was calculated. The addition of infliximab
to cultures of monocytes from patients with RA was
shown to decrease this ratio, corresponding to the
decreased IL-1␤ production in monocytes from patients
with RA following transmembrane TNF reverse signaling (mean ⫾ SEM 0.024 ⫾ 0.006 without infliximab
versus 0.008 ⫾ 0.001 with infliximab; P ⫽ 0.029), indicating that the effects of IL-1␤ were indeed inhibited. In
addition, concentrations of IL-6 and IL-8 in the culture
supernatants were determined. No significant influence
of infliximab on the secretion of those cytokines by
monocytes from patients with RA and healthy individuals was detected (data not shown).
Decreased spontaneous ex vivo apoptosis of
monocytes from patients with RA. Apoptosis of monocytes was investigated by double staining with annexin V
and the DNA dye PI, with subsequent analysis by flow
cytometry. A comparison of spontaneous monocyte ap-
Figure 3. Spontaneous and transmembrane TNF reverse signaling
induced–apoptosis in monocytes from patients with RA and control
subjects. A, Spontaneous apoptosis during in vitro culture, as evaluated
by annexin V and propidium iodide staining, in monocytes from
healthy donors (n ⫽ 50) and patients with RA (n ⫽ 48) that were
incubated for 16 hours in the presence of IgG. B, Representative dot
blots of annexin V– and propidium iodide–stained monocytes from
healthy donors and patients with RA after incubation with IgG or
infliximab. C, Apoptosis, as evaluated by annexin V and propidium
iodide staining, in monocytes from healthy donors (n ⫽ 50) and
patients with RA (n ⫽ 48) that were incubated for 16 hours in the
presence of infliximab or IgG. Data are presented as box plots, where
the boxes represent the 25th to 75th percentiles, the lines within the
boxes represent the median, and the lines outside the boxes represent
the 10th and 90th percentiles. D, Decreased transmembrane TNF
reverse signaling–induced apoptosis of monocytes from patients by
specific inhibition of casein kinase 1 (CK-1) activity using D4476.
Monocytes from patients with RA were incubated in the presence of
IgG or infliximab for 16 hours. Monocytes were preincubated with the
CK-1 inhibitor D4476 (n ⫽ 14). Bars in A and D show the mean and
SEM. See Figure 1 for other definitions.
2616
brane TNF, investigators claimed that induction of
apoptosis is the dominant event following transmembrane TNF reverse signaling (6,8,9). Because IL-1␤ is a
major survival factor for monocytes (12), the extent of
apoptosis triggered in vitro in monocytes from patients
with RA by the addition of infliximab and the resulting
IL-1␤ withdrawal was investigated. For that purpose,
monocytes from healthy donors and patients with RA
who had no history of receiving anti-TNF␣ therapy were
isolated from peripheral blood and incubated with 100
␮g/ml infliximab or control IgG for 16 hours. Apoptosis
was calculated as a ratio of the percentage of apoptotic
cells in infliximab-treated monocytes to that of IgGtreated monocytes.
Monocytes from healthy donors showed no increased apoptosis ratio after incubation with infliximab
(mean ⫾ SEM 1.06 ⫾ 0.04; n ⫽ 50), as shown in a
representative dot blot in Figure 3B. In contrast, transmembrane TNF reverse signaling induced apoptosis in
monocytes from patients with RA (mean ⫾ SEM apoptosis ratio 1.55 ⫾ 0.13; n ⫽ 48), as shown in a
representative dot blot in Figure 3B. At all earlier time
points analyzed (after 4 hours and 8 hours of incubation), no transmembrane TNF reverse signaling–
induced apoptosis was detectable (data not shown).
The DNA dye PI was used to differentiate between apoptotic cells and necrotic/late apoptotic cells. In
monocytes from patients with RA, transmembrane TNF
reverse signaling led to an increase in annexin V staining
in both PI-negative and PI-positive cells (mean ⫾ SEM
apoptosis ratio 1.55 ⫾ 0.13 and 1.25 ⫾ 0.05, respectively), although the increase was much lower in the
PI-positive cells. All further apoptosis ratios were calculated by using all annexin V–positive cells (PI-negative
and PI-positive cells).
When the ratios obtained for healthy donors and
patients with RA were compared, a significantly higher
ratio of induced apoptosis following transmembrane
TNF reverse signaling was evident in monocytes from
patients with RA (Figure 3C). This effect was also
noticed when etanercept was used instead of infliximab
(mean ⫾ SEM apoptosis ratio 1.86 ⫾ 0.28 versus 1.02 ⫾
0.05 [n ⫽ 11]; P ⫽ 0.005). However, this increase in the
rate of apoptosis reflected primarily the lower initial rate
of spontaneous apoptosis in monocytes from patients
with RA, while the absolute percentages of apoptotic
monocytes did not differ between infliximab-induced
apoptosis of monocytes from patients with RA and
spontaneous apoptosis of monocytes from control subjects (mean ⫾ SEM apoptosis 30.7 ⫾ 1.7% versus
32.44 ⫾ 2.0%; P ⫽ 0.51).
MEUSCH ET AL
Induction of apoptosis appeared to depend on
the expression of transmembrane TNF, because transmembrane TNF expression on monocytes from patients with RA correlated with the degree of
infliximab-induced apoptosis (R ⫽ 0.700, P ⫽ 0.016
[n ⫽ 11]). No such correlation was observed for
transmembrane TNF expression and apoptosis induction in monocytes from healthy donors (R ⫽ ⫺0.141,
P ⫽ 0.542 [n ⫽ 21]).
Importance of CK-1 activity for transmembrane
TNF reverse signaling–induced apoptosis. The CK-1
enzyme has been reported to be involved in proximal
signal transduction events of reverse signaling following
transmembrane TNF ligation. It is responsible for the
constitutive phosphorylation of at least 1 serine residue
in the cytoplasmic domain of transmembrane TNF, and
dephosphorylation of this serine residue by a stillunidentified phosphatase leads to calcium influx (13).
When monocytes from patients with RA were pretreated with 2 pan-phosphatase inhibitors, sodium vanadate and sodium fluoride, transmembrane TNF reverse
signaling–induced apoptosis was markedly reduced
(data not shown). Specific inhibition of CK-1 activity by
the selective inhibitor D4476 also led to a decrease in
transmembrane TNF reverse signaling–induced apoptosis of RA monocytes (Figure 3D).
No involvement of caspases in transmembrane
TNF reverse signaling–induced apoptosis. In contrast to
our findings, previous reports described infliximabinduced apoptosis in monocytes from healthy individuals
and a dependence on this process on the activation of
caspases 8, 9, and 3. Therefore, caspase activation in
monocytes from patients with RA was analyzed. Monocytes from healthy individuals were not included, because no apoptosis was observed. Under the culture
conditions used in the present study, caspases 8, 9, and 3
were not activated, as demonstrated by the absence of
cleavage of the inactive caspase pro forms (Figure 4A).
Accordingly, global inhibition of caspases by Z-VADFMK and inhibition of caspase 3 by Z-DEVD-FMK did
not influence transmembrane TNF reverse signaling–
induced apoptosis in monocytes (Figure 4B).
Other cellular events during apoptosis are cleavage of the DNA repair enzyme PARP-1 and degradation
of DNA. As shown in Figures 4C and D, no cleavage of
PARP-1 or DNA degradation was observed in transmembrane TNF reverse signaling–induced apoptosis. In
addition, expression of the antiapoptotic protein Bcl-xL
was not decreased in transmembrane TNF reverse
signaling–induced apoptosis (Figure 4C). In summary,
apoptosis following transmembrane TNF reverse signal-
EFFECTS OF ANTI-TNF␣ ANTIBODY ON MONOCYTES FROM PATIENTS WITH RA
Figure 4. Caspase-independent transmembrane tumor necrosis factor
(TNF) reverse signaling–induced apoptosis of monocytes from patients with rheumatoid arthritis (RA). Monocytes from patients with
RA were incubated in the presence of infliximab (inflix.) or IgG
control for 16 hours. A, Activity of caspase 8, caspase 9, and caspase 3,
as determined by immunoblot analysis. Arrows show pro forms and
cleaved caspases. Results are representative of 10 independent experiments. B, Influence of global inhibition of caspases by Z-VAD-FMK
and inhibition of caspase 3 by Z-DEVD-FMK on transmembrane TNF
reverse signaling–induced apoptosis in monocytes. Bars show the mean
and SEM. C, Expression of cleaved poly(ADP-ribose) polymerase 1
(PARP-1) and Bcl-xL, as determined by immunoblot analysis. Results
are representative of 3 independent experiments. D, DNA content, as
determined by propidium iodide staining. The marker is set on
subdiploid, apoptotic cells. Results are representative of 5 independent
experiments.
ing appears to be caspase independent and not accompanied by DNA fragmentation.
Inhibition of constitutive NF-␬B activation by
transmembrane TNF reverse signaling. One important
survival mechanism for monocytes and macrophages is
the constitutive activity of the transcription factor
NF-␬B (14–16), of which IL-1␤ is a strong activator (17).
To test whether constitutive NF-␬B activity is influenced
by reverse signaling of transmembrane TNF, the expression of the inhibitory protein I␬B␣ and its phosphorylation status were determined.
After 16 hours of culture, monocytes from patients with RA had low levels of I␬B␣ (Figure 5A),
which was highly phosphorylated (Figure 5B) and therefore subjected to ubiquitinylation and degradation. In
contrast, when monocytes from patients with RA were
cultured in the presence of infliximab, increased levels of
I␬B␣ were observed (see representative Western blot in
Figure 5A), which was poorly phosphorylated (Figure 5B).
High levels of I␬B␣ lead to the retention of
NF-␬B in the cytoplasm. To test whether the observed
2617
Figure 5. Transmembrane TNF reverse signaling–induced I␬B␣ accumulation and NF-␬B inhibition in monocytes from patients with RA.
Monocytes from patients with RA (n ⫽ 3) were incubated in the
presence of infliximab or IgG for 4 hours. A and B, Expression and
phosphorylation of I␬B␣, as determined by immunoblot analysis. Bars
show the mean and SEM. Immunoblots show 1 representative experiment. C, Results of electromobility shift assay. The gel shift was
present only with wild-type oligonucleotide (wt) and was almost absent
with mutant oligonucleotide (mt), demonstrating specificity of the
shifted band. Results are representative of 7 experiments. See Figure
4 for other definitions.
increase in I␬B␣ expression is indeed associated with
diminished translocation of NF-␬B into the nucleus,
nuclear extracts were prepared and subjected to a
NF-␬B electromobility shift assay. As shown in Figure
5C, NF-␬B was constitutively translocated into the nucleus in IgG-treated monocytes but was almost absent in
Figure 6. Prevention of anti-TNF–induced apoptosis of monocytes
from patients with RA by the addition of exogenous interleukin-1␤
(IL-1␤). Monocytes from patients with RA (n ⫽ 3) were incubated in
the presence of IgG, infliximab, or infliximab plus soluble IL-1␤ (100
pg/ml) for 16 hours. Bars show the mean and SEM. See Figure 4 for
other definitions.
2618
MEUSCH ET AL
infliximab-treated monocytes. The specificity of the observed NF-␬B:probe complex was verified by use of a
mutant probe that has a disrupted NF-␬B binding site.
Inhibition of transmembrane TNF reverse
signaling–induced apoptosis by the addition of IL-1␤.
To investigate the potential influence of IL-1␤ withdrawal on the rate of apoptosis, IL-1␤ was added to the
infliximab-treated monocyte cultures to simulate the
IL-1␤ levels in IgG-treated cultures. As shown in Figure
6, the addition of IL-1␤ completely reversed the increase
in transmembrane TNF reverse signaling–induced in
vitro apoptosis, again supporting the notion of an IL-1␤
deficit as the underlying mechanism of apoptosis induction in RA monocytes.
DISCUSSION
In the present study, we provide evidence demonstrating that monocytes from patients with RA express high amounts of transmembrane TNF and show
strong spontaneous in vitro IL-1␤ production. Triggering of reverse signaling of transmembrane TNF by
ligation with an anti-TNF antibody suppressed constitutive NF-␬B activation and inhibited this spontaneous,
constitutive production of IL-1␤, which in turn led to
increased in vitro apoptosis of RA monocytes.
Here, we demonstrate for the first time the
expression of transmembrane TNF on freshly isolated,
resting monocytes from patients with RA. The correlation between the observed transmembrane TNF reverse
signaling–triggered apoptosis in RA monocytes and the
determined transmembrane TNF expression levels indicates that transmembrane TNF reverse signaling is
indeed involved in apoptotic cell death in vitro. Inhibition of CK-1, an important kinase necessary for reverse
signaling of transmembrane TNF, led to a decrease in
infliximab-induced apoptosis, further pointing to transmembrane TNF reverse signaling as the mechanism
mediating infliximab-induced apoptosis.
Until now, transmembrane TNF reverse
signaling–induced apoptosis of monocytes from RA
patients has not been compared with that of monocytes
from healthy individuals, while several studies have
analyzed monocytes from patients with Crohn’s disease
(8). Only one other study investigating infliximabinduced apoptosis in monocytes from patients with RA
demonstrated increased annexin V expression after incubation with infliximab or etanercept, but this finding
did not reach statistical significance (7).
There have been studies on the ability of infliximab to induce apoptosis in vivo, mainly in the synovial
membrane of treated RA patients, but results have been
conflicting. Recently, Wijbrandts et al did not observe
any apoptosis in vivo after infliximab infusion (18),
whereas Catrina et al described increased synovial apoptosis, mainly in the monocyte/macrophage compartment (7). The network of proapoptotic and antiapoptotic factors in vivo is certainly more complex than that
in in vitro assays, however, and factors that are still
unknown might contribute to the observed discrepancies.
It has been shown in several studies that incubation of primary human monocytes from healthy donors
with infliximab leads to the induction of apoptosis
(6,8,9), although the opposite has also been reported
(19). We were unable to detect transmembrane TNF
reverse signaling–induced apoptosis in monocytes from
healthy individuals. Differences in the experimental
systems used and different monocyte purification methods (positive versus negative selection) might contribute
to the discrepant results. The demonstrated lack of
transmembrane TNF expression on the surface of monocytes from healthy control subjects can readily explain
the inability of infliximab to induce apoptosis, however.
This absence of transmembrane TNF expression on
resting monocytes has been reported previously (20,21),
although other reports have been conflicting (6,8,22),
again possibly due to the different purification methods
discussed above.
Monocytes undergo spontaneous apoptosis when
cultured in vitro (23,24). This spontaneous apoptosis can
be prevented by activation of monocytes with LPS or by
the addition of proinflammatory cytokines such as TNF
and IL-1␤ (12,23). Our results demonstrate that monocytes from patients with RA display a decreased level of
spontaneous apoptosis when cultured in vitro. Simultaneously, we observed spontaneous production of IL-1␤,
which was not detected in cultures of monocytes from
healthy individuals. Defects in macrophage apoptosis
have been observed previously in the synovial membrane
of patients with RA (25–27), but not in peripheral blood
monocytes (25). We observed the most significant difference in monocyte apoptosis between RA patients and
control subjects in the spontaneous apoptosis during in
vitro culture, which has not been previously reported. A
study involving Bim-deficient mice, which have more
severe disease, also suggested a possible pathogenetic
role for defective apoptosis in the exacerbation of inflammatory arthritis (28). Although an intrinsic defect
cannot be excluded as the underlying reason for the
decreased apoptosis, it appears equally possible that
predifferentiation or preactivation occurs in vivo in
EFFECTS OF ANTI-TNF␣ ANTIBODY ON MONOCYTES FROM PATIENTS WITH RA
patients with RA, which accounts for the increase in
transmembrane TNF and IL-1␤ and the decrease in
spontaneous apoptosis.
Due to the redundant functions of soluble and
transmembrane TNF, and due to the possibility of
forward and reverse signaling of transmembrane TNF
either via TNFR1/2 or via the cytoplasmic domain of
transmembrane TNF itself, we cannot entirely exclude
the inhibition of an autocrine effect of secreted TNF␣ by
infliximab as the underlying mechanism of apoptosis
induction in vitro. However, because CK-1 inhibited the
observed induction of apoptosis, which correlated
strongly with monocyte transmembrane TNF expression,
and because the addition of exogenous IL-1␤ reversed
the induction of apoptosis observed in patients with RA,
it appears unlikely that this neutralization of autocrine
TNF␣ is a major inductor of monocyte apoptosis in
vitro.
An inhibitory effect of reverse signaling via transmembrane TNF on LPS-induced IL-1␤ production has
already been reported for a monocytic cell line (4,29).
The published observation of decreased spontaneous ex
vivo IL-1␤ production of monocytes from patients with
RA 24 hours after infliximab infusion (30) also corroborates our finding that transmembrane TNF reverse
signaling inhibits spontaneous IL-1␤ production in RA.
The inhibition of apoptosis reported in this study was
reversed by addition of IL-1␤ to infliximab-treated
monocyte cultures, which again implies inhibition of the
survival factor IL-1␤ as a key mechanism in transmembrane TNF reverse signaling–induced apoptosis in RA
monocytes.
A similar mechanism has been described for
glucocorticoid-induced apoptosis in monocytes, with inhibition of IL-1␤ production by glucocorticoids and
prevention of glucocorticoid-induced apoptosis achieved
by the addition of IL-1␤ (31). Similarly, inhibition of
spontaneous apoptosis of neutrophils has been reported
to occur after the addition of IL-1␤ (32).
Several signal transduction mechanisms for transmembrane TNF reverse signaling–induced apoptosis
have been described. These include the activation of
caspase 8, caspase 9, and caspase 3, mitochondrial
release of cytochrome c, expression of the proapoptotic
proteins Bak and Bax, and PARP-1 cleavage in monocytes (6,8,9) and monocytic cell lines (33). To our
surprise, we were not able to observe activation of
caspase 8, caspase 9, and caspase 3, PARP-1 cleavage,
and degradation of DNA after incubation of monocytes
with infliximab. Furthermore, inhibition of caspases
did not prevent transmembrane TNF reverse
2619
signaling–induced apoptosis in monocytes from patients with RA.
Although the activation of caspases is the hallmark of apoptosis, reports on caspase-independent apoptosis have recently gained increasing attention (34–
36). Here, we demonstrate that the mechanism of
apoptosis induction following transmembrane TNF reverse signaling is largely caspase independent. We propose that the inhibition of constitutive NF-␬B activation
and subsequent suppression of IL-1␤ secretion is a
relevant mechanism of transmembrane TNF reverse
signaling–induced apoptosis.
The canonical NF-␬B activation pathway is primarily involved in de novo protein synthesis under
stimulatory conditions. However, NF-␬B is also constitutively active in many cell types, including monocytes
and macrophages (14–16), and has antiapoptotic effects
due to the induction of genes such as cellular inhibitors
of apoptosis, FLICE inhibitory protein, TNFRassociated factor 1 (TRAF1), and TRAF2 (37) in those
cells. Accordingly, inhibition of constitutive NF-␬B activation has been shown to induce apoptosis in a variety of
cell types under various conditions, including leukemia
cell lines (38–40), prostate cancer cells (41), multiple
myeloma cells (42), and mantle cell lymphoma cells (43),
but also human T lymphotropic virus type I–infected T
cell lines and primary adult T cell leukemia cells (44).
Our results demonstrated translocated NF-␬B in the
nucleus of resting monocytes from patients with RA and
a decrease in translocated NF-␬B after incubation of
monocytes with infliximab, while expression of I␬B␣
increased.
We hypothesize, therefore, that inhibition of
NF-␬B followed by caspase-independent induction of
apoptosis is an important signal transduction mechanism
event leading to the apoptotic signal elicited by transmembrane TNF reverse signaling. Interestingly, survival
of the monocytes could be restored by addition of IL-1␤,
pointing to an autocrine, NF-␬B–dependent survival
signal for monocytes that can be interrupted by transmembrane TNF reverse signaling. Accordingly, this induction of monocyte apoptosis could be an alternative
antiinflammatory mechanism of action of TNF inhibitors, with particular relevance at sites of monocyte
activation or monocytic cytokine production, such as the
rheumatoid synovial membrane. In addition, the observed inhibition of constitutive NF-␬B activation following transmembrane TNF reverse signaling is likely to
down-regulate the expression of NF-␬B–dependent
proinflammatory cytokines or chemokines, which might
2620
MEUSCH ET AL
contribute to the observed clinical effects of anti-TNF
treatment.
13.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Wagner had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Meusch, Rossol, Baerwald, Wagner.
Acquisition of data. Meusch, Rossol.
Analysis and interpretation of data. Meusch, Rossol, Baerwald, Hauschildt, Wagner.
14.
15.
16.
17.
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