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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3161-3168
CD137 Induces Proliferation and Endomitosis in Monocytes
By
Joachim Langstein,
Jan Michel, and
Herbert Schwarz
From the Department of Pathology, University of Regensburg,
Regensburg, Germany.
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ABSTRACT |
Peripheral monocytes are short-lived and are replenished from
hematopoietic stem cells whose proliferation is believed to be confined
to the bone marrow. Human peripheral monocytes are assumed not to be
able to proliferate. In this study we show that CD137 (ILA/4-1BB), a
member of the tumor necrosis factor receptor family, induces a
widespread and profound proliferation of human peripheral monocytes.
Macrophage colony-stimulating factor and granulocyte-macrophage
colony-stimulating factor are essential, but not sufficient for
proliferation. Additional soluble autocrine factors induced by CD137
are required. Induction of proliferation is mediated via reverse
signaling through a CD137 ligand, expressed constitutively by
peripheral monocytes. The ability of CD137 to induce proliferation in
human peripheral monocytes is not shared by any other known molecule.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CD137, ORIGINALLY NAMED 4-1BB and induced
by lymphocyte activation (ILA), is a tumor necrosis factor (TNF)
receptor family member and was identified in screens for receptors
expressed on activated lymphocytes.1-3 CD137 is expressed
by activated lymphocytes and monocytes and expression in primary cells
is strictly activation dependent.4 In several nonimmune
cells, expression is inducible by proinflammatory
cytokines.5,6 Soluble forms of CD137 are generated by
differential splicing and can be detected at enhanced concentrations in
sera of patients with rheumatoid arthritis.7 The gene for
human CD137 resides on chromosome 1p36, in a cluster of related
genes.8
Crosslinking of CD137 costimulates proliferation of T
lymphocytes,9-11 and CD137 ligand expressed by B
lymphocytes costimulates T-cell proliferation synergistically with
B7.12 Injected, agonistic anti-CD137 antibodies activate T
cells sufficiently for rejection of tumors and allogeneic transplants
in mice.13,14
While agonistic antibodies and the ligand to CD137 enhance lymphocyte
activation, CD137 protein has the opposite effect. It inhibits
proliferation of activated T lymphocytes and induces programmed cell
death. These T-cell inhibitory activities of CD137 require
immobilization of the protein, indicating transmission of a signal
through the ligand.11 Expression of the human CD137 ligand
is inducible in T lymphocytes and is constitutive in
monocytes.3
Reverse signaling through the CD137 ligand also takes place in
monocytes. Immobilized recombinant CD137 protein leads to activation of
the monocytes with enhanced expression of proinflammatory cytokines, inhibition of the anti-inflammatory cytokine IL-10, and induction of
activation markers such as intercellular adhesion
molecule-1 (ICAM).15 CD137 also prolongs monocyte survival
via induction of macrophage colony-stimulating factor
(M-CSF).16
Monocytes are key regulators of immune responses, essential for the
generation of beneficial antitumor and antipathogen immune reactions.
Monocytes also participate in the development of harmful autoimmune
diseases.17 They migrate from the circulation to sites of inflammations, attracted by signals such as chemokines, and
the accumulation of monocytes/macrophages is a characteristic feature
of chronic inflammation. This accumulation is further enhanced by
cytokines released at inflammatory sites, such as M-CSF,
granulocyte-macrophage CSF (GM-CSF), and interleukin-3 (IL-3), which
prolong the survival of monocytes/macrophages.18-20 The
accumulation of monocytes/macrophages at sites of inflammation has been
attributed so far solely to immigration and the extension of cell
survival, because human peripheral monocytes/macrophages have been
considered not to be able to proliferate.19,21
In this study we show that CD137 is able to induce a substantial degree
of proliferation/endomitosis in a large proportion of human peripheral
monocytes. This proliferative activity is not shared by M-CSF or any
other known molecule.
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MATERIALS AND METHODS |
Reagents.
M-CSF and neutralizing anti-M-CSF, anti-GM-CSF, and anti-IL-3
antibodies were obtained from R&D (Wiesbaden, Germany). Anti-M-CSF: clone 26730,11, protein A-purified IgG fraction of ascites fluid of
murine hybridoma; anti-GM-CSF: clone 3209.1, murine monoclonal, IgG1; and anti-IL-3: protein A-purified IgG fraction of
ascites fluid of murine hybridoma. Anti-HLA-DR was obtained from
Becton Dickinson (Heidelberg, Germany). The antibodies were free of
sodium azide. CD137-Fc protein was purchased from Alexis
(Grünberg, Germany). Human IgG1 Fc protein was
obtained from Accurate Chemical and Scientific Corp (Westbury, NY).
TNFRII-Fc protein was a gift from Daniela Maennel (University of Regensburg).
Cells and cell culture.
Human peripheral blood mononuclear cells (PBMCs) were isolated from
buffy coats of healthy volunteers. Buffy coats were diluted with 2 equal volumes of phosphate-buffered saline (PBS), overlaid onto an
equal volume of Histopaque (Sigma, Deisenhofen, Germany), and spun for
20 minutes at 1,200g. PBMCs that accumulated as a white layer
at the Percoll boundary were recovered and erythrocytes were lysed with
2 mL of 200 mmol/L NH4Cl, 10 mmol/L NaHCO3, and 10 mmol/L EDTA pH 7.4 for 2 minutes at room temperature. Cells were
washed 2 times with PBS, pelleted at 250g, and resuspended in
RPMI, 5% fetal calf serum (FCS). Primary monocytes were isolated by
elutriation.22 Elutriated monocytes were more than 95%
pure and contaminating T lymphocytes were less than 3% as estimated by
morphology and antigenic phenotype (CD14, CD3, CD4, and CD8 expression). Cells were cultured in polystyrene dishes (Becton Dickinson, Franklin Lakes, NJ) in RPMI 1640 supplemented with 5% FCS
at a concentration of 106/mL. The cell lines HL60 and
MonoMac were obtained from ATCC (Manassas, VA).
Enzyme-linked immunosorbent assay (ELISA).
ELISA kits were purchased from R&D Systems (Wiesbaden, Germany) and
performed according to the manufacturer's instructions. Cytokine
concentrations were determined in triplicate and are expressed as mean ± standard deviation.
Cell survival and apoptosis.
The number of living cells was determined by counting cells in 4 representative fields using an ocular with an engraved grid to ensure
equal sizes of the evaluated fields. Dead and live cells were
distinguished by trypan blue exclusion.
Apoptosis was determined via DNA fragmentation by the `Cell Death
Detection ELISA Plus' (Boehringer Mannheim, Mannheim, Germany) following instructions provided by the manufacturer. Measurements were
performed in triplicate.
Cell proliferation.
For measurement of proliferation of individual cells the `In situ
proliferation kit' (Boehringer Mannheim) was used. A total of 3 × 105 cells were seeded per chamber of an 8-chamber
slide (Falcon; Becton Dickinson, Heidelberg, Germany), coated with Fc
or CD137-Fc protein and grown for 10 days. Bromodeoxyuridine (BrdU), 10 µmol/L, was added for 60 minutes. Incorporated BrdU was visualized
according to kit instructions, by staining with mouse anti-BrdU and
sheep anti-mouse-fluorescein isothiocyanate (FITC). Monocytes were
identified by staining with a phycoerythrin-labeled, anti-CD14 antibody
(2 µg/mL; Immunotech, Marseille, France). Chromatin was stained by 5 µg/mL Hoechst 33342 (Sigma, Deisenhofen, Germany) for 5 minutes.
Proliferation of cell populations was determined in 96-well microtiter
plates. 105 monocytes per well were pulsed for 24 hours
with 0.5 µCi 3H-thymidine, harvested, and evaluated on
the TopCount microplate scintillation counter (Packard,
Meriden, CT). Each condition was performed in triplicate and results
are depicted as means ± standard deviation.
Statistical analysis.
Statistical significance was evaluated by the Mann Whitney U test using
SPSS software (SPSS Inc, Chicago, IL).
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RESULTS |
CD137 induces apoptosis in monocytes.
CD137 has been shown to substantially prolong the survival of human
primary peripheral monocytes.16 To investigate the
mechanism of this activity, we tested whether CD137 prevents apoptotic
cell death. Monocytes were cultured on tissue culture dishes coated with a fusion protein consisting of the extracellular domain of CD137
and the constant domain of human immunglobulin G1 (Fc). Coating was
performed with a solution of 1 µg/mL protein in PBS at 4°C
overnight. Untreated and Fc protein-coated plates were used as
controls. Monocytes in these control groups died due to lack of
stimulation and/or survival signals.23-25 In the CD137-Fc protein-coated wells, the number of living monocytes was significantly higher from day 5 of culture onward
(Fig 1).
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| Fig 1.
Induction of monocyte apoptosis by CD137. 105
primary monocytes were cultured on immobilized Fc or CD137-Fc protein
or on untreated plates (control). The extent of apoptosis (top) and the
number of living cells, determined by trypan blue exclusion (bottom)
were evaluated at days 1, 3, 5, and 7. Error bars indicate standard
deviation. The differences in rates of apoptosis and the number of
living cells between Fc and CD137-Fc treated cultures were significant
starting at days 3 and 5, respectively, with P < .05. Comparable results were obtained in 3 separate experiments.
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The extent of apoptosis in the same cultures was quantified by
measuring the amount of fragmented DNA (Fig 1). Quite unexpectedly, we
found a higher degree of apoptosis in monocyte cultures treated with
CD137 protein, compared to untreated cultures or cultures treated with
the Fc control protein.
CD137 induces proliferation of peripheral monocytes.
Despite induction of apoptosis by CD137, the fact remained that CD137
prolonged the survival of the monocyte population. These seemingly
contradictory results could best be reconciled by the assumption that
CD137 induces proliferation of monocytes, compensating for the loss of
cells through apoptosis. As measured by 3H-thymidine
incorporation, CD137 induced, in fact, a strong proliferation of
monocytes (Fig 2A). Proliferation
correlated positively with the time monocytes were grown in the
presence of CD137 protein and reached maximum levels between day 7 to
10, with a 30-fold or even higher increase in 3H-thymidine
incorporation compared with control cells. There was no difference
between monocytes in untreated and Fc protein-coated wells (not shown).
Also, in the CD137-Fc-treated monocyte cultures, but not in control
cultures, colonies or cell aggregates were observed, which could wholly
or partly result from proliferating monocytes (Fig 2B).
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| Fig 2.
CD137 induces proliferation of peripheral monocytes. (A)
105 monocytes were cultured on 96-well plates coated with
Fc or CD137-Fc protein. Proliferation was determined daily by a 24-hour
pulse with 0.5 µCi 3H-thymidine and differed
significantly from day 3 onward, with P < .02. (B)
Colonies/aggregates formed on CD137-treated monocytes. Photographs were
taken at day 10 of culture at a magnification of 200×.
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In line with the substantial increase in 3H-thymidine
incorporation is that CD137-induced proliferation was rather
wide-spread. Upon labeling CD137 treated monocytes with BrdU for only 1 hour, and subsequent detection with fluoresceine-labeled, anti-BrdU antibodies, 9.3% of the cells (55 ± 6 of 589 ± 43)
were found to replicate their DNA (Fig 3A).
Peripheral monocytes grown on Fc-coated dishes did not show
incorporation of BrdU (not shown).
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| Fig 3.
Verification of proliferating cells as monocytes. (A)
Monocytes were grown for 8 days with immobilized CD137 protein and
labeled for 60 minutes with 10 µmol/L BrdU (yellow). (B) Monocytes
were cultured on immobilized CD137-Fc protein on chamber slides. After
10 days the cells were labeled for 60 minutes with 10 µmol/L BrdU
(yellow). Monocytes were identified by staining for CD14 (red), and
nuclei were visualized by Hoechst 33342 (blue). Similar results were
obtained in 3 independent experiments for (A) and (B), respectively.
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We confirmed by immunocytochemistry that the proliferating cells were
in fact monocytes and not other blood cells. Proliferation was
determined by BrdU-incorporation and the identity of monocytes was
verified by simultaneously staining for CD14, a cell surface protein
specific to monocytic cells. Nuclei were visualized by staining with
the DNA intercalating dye Hoechst 33342. As shown in Fig 3B, the
proliferating cells have features characteristic of monocytes: they are
CD14-positive, are multi-nucleated, and have the morphological
appearance of monocytes/macrophages.
Other molecules that also bind to constitutively expressed molecules on
monocytes, like TNFRII-Fc or anti-HLA-DR were not able to induce
proliferation (Fig 4). Furthermore, CD137
had no effect on proliferation of the monocytic cell lines HL60 and
MonoMac (Table 1), nor did it induce
adherence and spreading of these nonadherent cells (not shown).
However, the spontaneous rate of proliferation of the two lines was
more than an order of magnitude higher than the rate of primary
monocytes that were stimulated with CD137 for 8 days (Table
1).
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| Fig 4.
Specificity of CD137-induced monocyte proliferation.
105 peripheral monocytes were cultured on plates coated
with 1 µg/mL of Fc, CD137-Fc protein, anti-HLA-DR antibody, or a
fusion protein consisting of the extracellular domain of the type 2 TNF
receptor and the constant domain of immunoglobulin G1 (TNFRII-Fc).
Proliferation was determined at day 8 by 3H-thymidine
incorporation and comparable results were obtained in 2 independent
experiments.
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M-CSF and GM-CSF are essential but not sufficient for CD137-induced
monocyte proliferation.
Because M-CSF is an essential monocyte survival
factor,26,27 and because CD137 prolongs survival of
monocytes via induction of M-CSF,16 it is conceivable that
M-CSF would also be required for CD137-induced proliferation of
monocytes. Confirming this hypothesis, neutralizing anti-M-CSF
antibodies reduced CD137 induced proliferation almost completely
(Fig 5A).
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| Fig 5.
M-CSF and GM-CSF are essential, but not sufficient, for
CD137-induced monocyte proliferation. (A) 105 peripheral
monocytes were cultured on immobilized Fc or CD137-Fc protein.
Neutralizing anti-M-CSF antibody (2 µg/mL), anti-GM-CSF antibody (2 µg/mL), and anti-IL-3 antibody (2 µg/mL) or isotype control
(IgG2a; 2 µg/mL) were added where indicated. Proliferation was
determined at day 10 by 3H-thymidine incorporation in
triplicate conditions. (B) M-CSF and GM-CSF do not induce proliferation
of monocytes. Peripheral monocytes were cultured on 96-well plates
coated with Fc or CD137-Fc protein (1 µg/mL) or M-CSF and
GM-CSF at indicated concentrations (ng/mL). Proliferation was
determined at day 10 by 3H-thymidine incorporation. Similar
results were obtained in 3 separate experiments for (A) and
(B).
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Besides M-CSF, GM-CSF and IL-3 have been reported to be important for
monocyte survival.18 Complete inhibition of monocyte proliferation was achieved by neutralizing M-CSF and GM-CSF.
Neutralizing GM-CSF or IL-3 alone had only a slight (18% reduction) or
no effect on CD137-induced proliferation, respectively. The
sequestration of both GM-CSF and IL-3 together synergistically reduced
proliferation to one third (Fig 5A).
These experiments established M-CSF and GM-CSF as being essential for
CD137-induced proliferation of peripheral monocytes. However, they were
not sufficient. M-CSF and GM-CSF at concentrations induced by CD137
elicited only a negligible proliferation (Fig 5B). M-CSF is induced by
CD137 up to 10 ng/mL,16 whereas no GM-CSF could be measured
(not shown). In vivo, M-CSF and GM-CSF are detectable around 10 ng/mL
and 50 pg/mL, respectively.28-30 Even at significantly
higher concentrations, ie, at 100 and 1 ng/mL, respectively, M-CSF and
GM-CSF induced only a fraction of the proliferation observed with CD137
(Fig 5B). This may point to the existence of other, additional factors
that are induced by CD137 in monocytes, and that contribute in an
autocrine fashion to CD137-induced proliferation of monocytes.
CD137-induced monocyte proliferation is mediated by soluble autocrine
factor(s).
Next, we wanted to evaluate whether these additional factors required
for monocyte proliferation were soluble or cell-surface-bound. Supernatants of monocytes cultured for 24 hours on immobilized CD137
protein were harvested and cells were removed by centrifugation for 5 minutes at 12,000g. Transfer of this conditioned medium to
untreated monocytes from the same donor dose-dependently induced cell
growth (Fig 6A) and proliferation (Fig 6B).
Addition of neutralizing anti-M-CSF antibodies (2 µg/mL) to
conditioned medium blocked induction of cell growth and proliferation
(not shown).
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| Fig 6.
CD137 induces proliferation of monocytes via autocrine
induction of soluble factor(s). 105 peripheral monocytes
were cultured on immobilized Fc or CD137-Fc protein for 24 hours. 0, 10, 20, or 30 µL of conditioned supernatant of these cultures were
transferred to 100 µL of new cultures with untreated monocytes. (A)
After 8 days, the cells were photographed at a magnification of 300×.
The bottom panel depicts monocytes grown on immobilized CD137-Fc
protein for the 8 days. (B) Proliferation of the cultures from (A) was
determined at day 8 by a 24-hour pulse with 0.5 µCi
3H-thymidine. ( ) Proliferation of monocytes on
immobilized CD137-Fc protein. Cultures supplemented with conditioned
supernatant from CD137-Fc-activated monocytes proliferated
significantly stronger; P < .007. This experiment was
repeated 3 times with similar results.
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Immobilization of CD137 protein is necessary for monocyte
proliferation.
Induction of monocyte proliferation by CD137 required immobilization of
the CD137 protein, eg, by coating of the protein to the surface of the
tissue culture dishes (Fig 7). If CD137 was given as a soluble protein, after preventing its immobilization by
prior coating of the dishes unspecifically with bovine serum albumin,
the proliferative activity of CD137 was absent. Therefore, induction of
monocyte proliferation by CD137 seems to be mediated by crosslinking of
a CD137 ligand expressed by monocytes.
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| Fig 7.
Immobilized, but not soluble, CD137 induces monocyte
proliferation. 105 peripheral monocytes were cultured on
immobilized Fc or CD137-Fc protein. Proliferation was determined at day
10 by 3H-thymidine incorporation. Identical results were
obtained in 3 independent experiments.
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DISCUSSION |
Based on the survival enhancing activity of CD137,16 it was
quite surprising to find a higher rate of apoptosis in CD137-treated monocytes. Considering that CD137 causes activation in
monocytes,15 this situation resembles very much the
phenomenon of activation induced cell death, originally described for
lymphocytes. Cell activators that induce activation and proliferation
simultaneously induce apoptosis in a certain percentage of the cells,
but as long as proliferation outweighs cell death, the cell population expands.31
The contradictory results of the simultaneous induction of cell
survival and apoptosis led us to test whether CD137 is able to induce
proliferation of monocytes. The finding that human peripheral monocytes
in fact do proliferate came as a surprise. The generally held opinion
is that monocytes originate from hematopoietic stem cells, the
proliferation of which is confined to the bone marrow. Peripheral
monocytes and macrophages are assumed not to be able to
proliferate.19,21 This notion was supported by experiments measuring only marginal proliferation of peripheral monocytes, even
after treatment with monocyte activators and survival factors. None or
only 0.3% of peripheral monocytes were found to undergo DNA
replication in response to M-CSF and GM-CSF during a 20-hour labeling
period.19,25 This low percentage of proliferating cells was
in line with no increase or an increase by only a factor of 2 in
incorporation of 3H-thymidine induced by
M-CSF.19,25 Our results confirm these earlier findings. But
in contrast to M-CSF, CD137 induced a strong proliferation in a large
proportion of the cells. Upon labeling for only 1 hour, 9.3% of the
cells had incorporated bromodeoxyuridine into their DNA, implying that
a substantial percentage of the cells were proliferating. Furthermore,
3H-thymidine incorporation at its maximum was 30- to
80-fold higher in CD137-treated monocytes compared with control cells.
By demonstrating that the DNA replicating cells were CD14-positive, had
multiple nuclei, and the morphological appearance of monocytes, we
could show unequivocally that the proliferating cells were in fact monocytes.
Further, CD137 has been shown to inhibit proliferation and to induce
apoptosis in T lymphocytes.11 Therefore, these cells would
not, even if present, be able to contribute to the observed proliferation.
Endomitosis and cell fusion have been shown to occur in
monocytes.32,33 Proliferation of monocytes induced by CD137
likely results, to some extent, in endomitosis. Many polynucleated
cells are observed in CD137-protein-treated monocyte cultures. There are two indications that they arise at least partly from endomitosis rather than solely from cell fusion: First, replicating nuclei are
present in already polynucleated cells that do not show signs of cell
division. Second, a decrease in the cell number, as would be expected
with cell fusion, is not observed. The few small BrdU-positive cells
present in CD137-treated cultures may represent monocytes that just
have undergone cell division. Generally, the simultaneously ongoing
processes of proliferation, endomitosis, apoptosis, and cell fusion
make it impossible to quantify the number of newly emerging and dying
cells. The fact that the rate of proliferation of the monocytic cell
lines was more than an order of magnitude larger than that of the
primary cells may be due to an in vitro selection for rapid growth and
an acquisition of mutations further supporting a high proliferation
rate.34,35
For monocyte survival, M-CSF has been reported to be essential and
sufficient.26,27 For proliferation of monocytes, M-CSF is
also essential because sequestration of M-CSF is able to abolish proliferation almost completely. However, M-CSF is not sufficient, because at physiological and even at higher concentrations it is not
able to induce proliferation of monocytes on its own. Also, the
combination of M-CSF and GM-CSF failed to replicate the proliferative activity of CD137. Therefore, additional factor(s) contributing to
monocyte proliferation must exist.
The ability of conditioned medium from CD137-treated monocytes to
induce adherence, spreading, and proliferation in other monocytes
proves (1) the soluble, and (2) the autocrine nature of these
additional factor(s). Adherence and spreading can be induced by M-CSF,
which is upregulated by CD137, and sequestration of M-CSF by
neutralizing antibodies proves that M-CSF is also an essential factor
in the conditioned medium. But M-CSF did not induce proliferation.
Therefore, these additional factors may be novel cytokines or a
combination of M-CSF and known cytokines.
Although significant proliferation of monocytes in isolation has not
been demonstrated before, the concept that peripheral monocytes are
incapable of proliferating has already been questioned by two recent
studies. Local proliferation of peripheral monocytes in vivo, in a
granuloma, a specialized inflammatory reaction, and in vitro, after
coculturing of monocytes with aortic endothelial cells, has been
reported.36,37 In both systems M-CSF was essential but not
sufficient for proliferation of monocytes, a situation identical with
CD137-induced monocyte proliferation. Since CD137 is expressed at sites
of inflammation,4 and can be expressed by endothelial cells
(H.S., unpublished observation, March 1997), CD137
could have been the factor enabling monocyte proliferation in these
systems. Generally, it would be sites of inflammation, where CD137 is
expressed and could induce activation and proliferation of monocytes,
and thereby enhance inflammatory reactions.
It needs to be emphasized that CD137 is a cell surface receptor and the
activities described here are mediated by crosslinking of a CD137
ligand and take place in the ligand-bearing monocyte. Dimerization of
CD137 ligand is not sufficient to induce proliferation, because the
CD137-Fc protein used in these studies dimerizes via its Fc domains and
the soluble dimeric CD137 protein is not able to induce monocyte
proliferation. Higher-order multimerization of the ligand achieved by
immobilization of the CD137 protein to the tissue culture platesis
required. Transduction of a signal through a CD137 ligand is also
responsible for activation of monocytes and for inhibition of
proliferation and induction of apoptosis in T
lymphocytes.11,15 Because, in the TNF receptor and ligand families, the receptors as well as the ligands are membrane-bound molecules, a bidirectional transduction of signals is possible. Bidirectional transduction of signals through the receptor as well as
the ligand has also been described for the OX40, CD40, and CD30
receptor/ligand systems, three other members of the TNF receptor and
ligand families.38-41 A CD137 ligand has been isolated that
is expressed constitutively by monocytes,3 thus enabling resting monocytes to respond to CD137 protein.
The proliferative activity of CD137 on peripheral monocytes is not
shared by other molecules binding to cell surface proteins on
monocytes, nor by well-established monocyte growth factors, like M-CSF,
GM-CSF, nor any other known molecule, and constitutes an activity so
far unique to CD137. Given the central position of monocytes in the
regulation of immune responses, inhibition of CD137 may represent a new
way to interfere with destructive autoimmune reactions. Accordingly,
protective immune responses against pathogens and malignant cells may
be enhanced by CD137-mediated activation of monocytic and dendritic
cells, cells that are intensively studied for immune therapy for
cancer.42-44
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ACKNOWLEDGMENT |
We thank Gitte Krause for excellent technical assistance, and M. Kreutz
and R. Andreesen for peripheral monocytes.
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FOOTNOTES |
Submitted September 15, 1998; accepted June 24, 1999.
Supported by the Deutsche Forschungsgemeinschaft.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Herbert Schwarz, Department of Pathology,
University of Regensburg, 93042 Regensburg, Germany.
| |
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