CD137 (ILA/4-1BB), expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes
Georg Kienzle1,2 and
Johannes von Kempis1
1 Division of Rheumatology and Clinical Immunology, University Hospital Department of Medicine, Hugstetter Strasse 55, 79106 Freiburg, Germany
2 Department of Biology, University of Freiburg, Schaentzlestrasse 1, 79104 Freiburg, Germany
Correspondence to:
J. von Kempis
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Abstract
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Human CD137 is a member of the tumor necrosis factor (TNF) receptor family and the homologue of murine 4-1BB. Recent studies have demonstrated that CD137 promotes accessory T cell activation, and regulates proliferation and survival of T lymphocytes. This study reports on the expression and function of CD137 in peripheral blood monocytes. While monocytes showed constitutive expression in 10 out of 18 healthy donors, CD137 was not expressed on resting T or B lymphocytes. Immobilized antibodies to CD137 markedly induced the production of IL-8 and TNF-
protein and mRNA, and led to inhibition of IL-10 expression by primary monocytes. Furthermore, cross-linking of CD137 on monocytes resulted in an increase of B lymphocyte apoptosis mediated by direct cellcell contact of both cell populations. In conclusion, this study identified CD137 as a new receptor involved in monocyte activation by inducing a characteristic cytokine release profile. In addition, CD137 may play a role in monocyte-dependent control of B lymphocyte survival.
Keywords: apoptosis, cellular activation, human, inflammatory mediators, monocytes/macrophages
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Introduction
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CD137 is a member of the tumor necrosis factor (TNF) receptor family and the human homologue of mouse 4-1BB (1). Similar to its mouse counterpart, human CD137 was first described as a gene induced by lymphocyte activation (ILA) (2). Meanwhile, 4-1BB and ILA have been designated CD137 (3). As with other members of the TNF receptor family, human CD137 is located on chromosome 1p36 (4).
The TNF receptor family consists of a growing number of type I transmembrane proteins such as TNF receptor I and II, the low-affinity nerve growth factor receptor, CD27, CD30, CD40, CD95, DR-3, DR-4, DR-5, DR-6, OX-40, and some viral homologues (510) which are characterized by cysteine-rich repeats in the extracellular part. Most of these proteins are involved in the regulation of activation and/or survival of cells (9). Many ligands for these receptors, among them the ligands for mouse and human CD137 (11,12), have been cloned and identify a family of mostly surface-bound type II transmembrane proteins with structural homology (13).
Human CD137 is present in immune cells such as activated T and B lymphocytes and monocytes, and a number of other cell lineages (14). In addition, it is expressed in cells of mesenchymal origin in a differentiation-dependent and stimulus-specific manner (15). CD137 is a potent co- stimulatory molecule to T lymphocytes in both man and mouse (16,17). Co-stimulation of T cells by CD137 has been reported to act dependently and independently of CD28 (18,19). In addition, CD137 regulates survival of T lymphocytes (17,20) and amplifies tumor immunity in mice (21,22). The biological functions of human and mouse CD137 may differ since only the murine protein binds to laminin in addition to the specific ligand (23). Recently, the association and usage of TNF receptor-associated factors (TRAF) for signaling has been shown for both human and mouse CD137. For both species, signal transduction via CD137 resulted in the activation of NF
B (2426).
Monocytes and macrophages belong to the main effector cells of the immune system, being engaged in host defense mechanisms against pathogens and tumor cells or acting as antigen-presenting cells (27). Monocytes are known as a potent source of multiple biologically active molecules, such as enzymes, plasma proteins and cytokines (28).
The objective of the present study was to determine whether CD137 is expressed in peripheral blood cells. It is shown that CD137 is expressed by monocytes, but not by lymphocytes and is further induced during cell culture. Cross-linking of CD137 leads to monocyte activation as demonstrated by increased release of IL-8 and TNF-
, and by inhibition of IL-10 production. Signaling through CD137 induces apoptosis of B lymphocytes by mechanisms requiring direct cellcell contact between monocytes and B cells.
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Methods
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Cell isolation and culture
Human peripheral blood mononuclear cells (PBMC) were prepared by Ficoll density gradient sedimentation according to standard protocols. Isolated cells were used either directly for flow cytometry analysis and isolation of total RNA or cultured in RPMI 1640 medium supplemented with L-glutamine, antibiotics and 10% FBS.
Flow cytometry
For flow cytometry, cells (24x105 cells each) were incubated with phycoerythrin (PE)-labeled specific mAb to CD4 (MT310; Dako, Glostrup, Denmark), CD19 (HD37; Dako), CD14 (Tuek4; Caltag, San Francisco, CA) and CD8 (3B5; Caltag). For detection of CD137 the unlabeled antibody M131, kindly provided by Immunex (Seattle, WA) was used. This antibody and unlabeled MOPC21 (Sigma, St Louis, MO), as isotype control, were detected by FITC-labeled goat anti-mouse secondary antibodies. Cells were washed with staining buffer (PBS, 3% FBS and 0.1% sodium azide), incubated with propidium iodide (PI; 1 µg/ml) and analyzed on a FACScan (Becton Dickinson, Mountain View, CA). The software program CellQuest 3.0.1 (Becton Dickinson) was used on a FACScan. The analysis of CD137 expression in subpopulations of PBMC was performed after gating for viable cells by forward and side scatter and after exclusion of PI.
Purification of monocytes and B lymphocytes
For the enrichment of monocytes from Ficoll-isolated PBMC, an antibody-mediated depletion technique was used (MACS Monocyte Isolation Kit; Miltenyi, Bergisch Gladbach, Germany). Using a cocktail of MicroBeads-coupled antibodies, lymphocytes, basophils, dendritic and NK cells were removed. Isolated monocytes had an average purity of 96%, as analyzed by forward/side scatter and CD14 staining on flow cytometry. B cells were isolated by CD19Dynabeads (Dynal, Hamburg, Germany) and magnetic separation of labeled cells with subsequent detachment of CD19 antibodies, as provided by the manufacturer. Isolated cells were of >97% CD19+ on flow cytometry.
Complete depletion of monocytes was achieved by staining of PBMC with CD14Dynabeads (Dynal) and removal of magnetic-labeled cells, according to the protocol provided by the manufacturer.
Stimulation of monocytes by antibodies
Twenty-four-well Nunclon tissue culture plates (Nunc, Roskilde, Denmark) were precoated with 200 µl of antibody dilution (5 µg/ml in PBS) for 3 h at 37°C. After incubation, wells were washed with PBS and 1.5x106 PBMC or 1x106 purified monocytes were added to culture plates. Cells were incubated at 37°C and 5% CO2.
In parallel, after 1.5 h of culture on immobilized antibodies, non-adherent cells of PBMC were removed by repeated (3 times) vortexing of the whole-tissue culture plate and change of media. Remaining cells were lysed for RNA isolation followed by cDNA synthesis and quantitative PCR for cytokine expression.
Immunohistochemistry
Isolated monocytes were cultured on poly-L-lysinecoated glass slides, fixed by methanol-containing fixative (Hemacolor; Merck, Darmstadt, Germany), and stained with antibodies to CD137, CD68 and isotype control antibody MOPC21. Endogenous peroxidase activity was blocked by phenylhydrazine (0.1% in PBS) and unspecific binding by horse serum (5% in PBS). Primary antibodies were detected by biotinylated horse anti-mouse-IgG and streptavidin peroxidase.
Cytokine ELISA
The production of cytokines was determined in culture supernatants by cytokine-specific ELISA. For IL-8 and IL-10, the Compact ELISA kit from CLB (Amsterdam, The Netherlands) and for TNF-
a matched antibody pair from PharMingen (Hamburg, Germany) were used according to the manufacturer's instructions.
RNA isolation
Total RNA was isolated by the RNeasy isolation kit (Quiagen, Hilden, Germany) according to the manufacturer's instructions. Cells were lysed directly in culture plates and isolated RNA was resuspended in RNase-free water and stored at 70°C until used in RT-PCR.
Quantitation of cytokine transcripts
The level of transcripts for IL-8 and TNF-
was determined by competitive PCR after reverse transcription of total RNA with random hexamer primers as described (29). We used the multispecific competitor vector pQA1 (30) for quantitative PCR of IL-8 and TNF-
. To standardize the amount of cDNA, quantitative PCR reactions for ß2-microglobulin were performed for each sample. Briefly, PCR reactions with equal aliquots of the diluted reverse transcription reaction containing ~40 ng cDNA equivalents and cytokine specific primers were added to serial 4-fold dilutions of known molarity (5x106 to 7.5x101 molecules) of the competitor. During co-amplification reactions, primers compete for annealing and amplification of both competitor and cDNA resulting in PCR products easily separated by agarose gel electrophoresis. Equal band intensities of both fragments allow the quantitation of cDNA transcripts relative to the competitor on densitometry.
Analysis of cell-death/apoptosis
Cell death of lymphocytes was analyzed by the ability of intact, viable cells to exclude PI. PI accumulates in dead cells and can be demonstrated on flow cytometry. Cultured PBMC were stained for CD19 to identify B cells and incubated with PI (1 µg/ml) for 10 min on ice. Cells were analyzed on single-cell levels by flow cytometry. In addition, cell death was determined by reduced size and increased granularity of cells as analyzed by forward and side scatter on flow cytometry. Demonstration of apoptosis was done by the enzymemediated incorporation of FITC-labeled nucleotides at DNA strand breaks (TUNEL). The In Situ Cell Death Detection kit (Boehringer Mannheim, Mannheim, Germany) was used according to the manufacturers instructions.
Reagents and other materials
Endotoxin content of RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 10% FBS was 0.06 ng/ml. For stimulation of monocytes, antibody 4B4-1 to CD137 (Alexis, Gruenberg, Germany) and MOPC21 (Sigma) as isotype control were used. A fusion protein of the extracellular part of CD137 and the human IgG1 Fc terminus (CD137Ig) was purchased from Alexis. The mAb M121 (msIgG1) to CD137, used for inhibition of the agonistic effects of mAb 4B4-1, as well as M131 were kindly provided by Immunex. The antibody Nok-1 against the Fas ligand was purchased from PharMingen free of azide and with low endotoxin (Hamburg, Germany). The antibody Ki-M6 (CD68) from BMA (Augst, Switzerland) was used for immunhistochemistry. For culture of monocytes with minimized adherence of cells, Petriperm Teflon vials (Haraeus, Osterode, Germany) or Teflon bottles (Nalgene, Rochester, NY) were used. Transwell membranes (Costar, Bodenheim, Germany) with a pore size of 0.4 µm together with 24-well tissue culture plates were taken for co-cultures of cell fractions without direct cellular contact.
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Results
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Expression of CD137 in human primary monocytes
Expression of CD137 in different cell types is known to be dependent on activation by the specific T or B cell receptor, or by proinflammatory cytokines (14-16). Earlier findings indicated that this is also true for human primary monocytes where expression of CD137 mRNA was only found after induction by stimuli such as phorbol esters and IL-1 (14). A first set of experiments in this study, however, demonstrated that monocytes can constitutively express CD137. CD137 protein expression was analyzed by flow cytometry of mononuclear cells directly after isolation from the peripheral blood of healthy donors. In 10 out of 18 donors, constitutive expression of CD137 on monocytes, ranging from 5 to 72%, was observed, whereas in all 18 samples we failed to detect CD137 in resting T or B cells (not shown). Time course experiments of monocytes on different surfaces, such as Teflon (Fig. 1A
) or glass (Fig. 1B and C
), showed that CD137 is immediately up-regulated after beginning of cell cultures, presumably due to contact-mediated mechanisms. After culture of 448 h, very high expression on most monocytes was seen (Fig. 1A
) which was not further regulated by IL-1 or TNF-
(not shown). To exclude unspecific induction of CD137 on monocytes by isolation procedures, expression was also analyzed in untreated peripheral blood, without density-gradient sedimentation, confirming constitutive expression of the receptor on monocytes (not shown).

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Fig. 1. Cultured monocytes in vitro express high levels of CD137 protein. Monocytes were isolated from PBMC by depletion of non-monocytic cells using antibodies coupled to magnetic beads. Purity of the isolated cell population was controlled by flow cytometry. (A) Analysis of CD137 expression by monocytes cultured in Teflon vials. Monocytes were stained for CD137 with mAb M131 (2) or isotype control (1) directly after isolation and analyzed by flow cytometry. Staining for CD137 was also performed after 4 h (3), 16 h (4) and 48 h (5) of culture. One representative experiment out of three is shown. This rapid induction of CD137 expression in culture was also observed in monocytes not constitutively expressing CD137 directly after isolation. (B) The majority of monocytes cultured for 5 h on glass slides and subjected to immunohistochemistry with mAb M131 showed strong staining for CD137 (original magnification x400). (C) Control experiments performed in parallel with isotype control antibodies did not show staining of cells (original magnification x400). Immunhistochemistry of CD137 expression was performed 4 times with identical results. Staining of cells by the antibody M131 could be completely inhibited by preincubation of the antibody with an CD137Ig fusion protein but not with human Fc alone (not shown).
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Induction of cytokine mRNA in primary monocytes by CD137
To address the role of CD137 in monocyte function, monocytes derived from peripheral blood were cultured in the presence of antibodies to CD137 and analyzed for cytokine expression. The purity of the isolated cell population was confirmed by morphology and staining for CD68 identifying 82% of adherent cells as monocytes. The amount of mRNA/cDNA transcripts for TNF-
and IL-8 was determined by quantitative PCR, using a multispecific internal plasmid competitor. Stimulation of monocytes via CD137 resulted in a 4-fold up-regulation of cDNA products of both cytokines, TNF-
and IL-8, compared to control antibodies (Fig. 2
). The equivalence point (1:1 ratio) of target to competitor molecules for TNF-
was reached at 2x104 competitor molecules and was induced to 7.8x104 molecules/40 ng cDNA by antibodies to CD137 as determined by densitometric analysis. For IL-8, signaling through CD137 induced 6.2x105 molecules as compared to 1.56x105 molecules in the controls.
Regulation of cytokine protein expression by CD137 signaling
In addition to mRNA levels, we further analyzed the regulation of cytokine protein synthesis by CD137. PBMC from healthy donors were isolated and cultured in plastic dishes with immobilized mAb to CD137 or with control antibodies. Flow cytometry at the beginning, during and at the end of culture revealed CD14+ monocytes as the only cell population expressing CD137. This was further confirmed by immunohistochemistry with antibodies to CD137 and CD68 (not shown). Supernatants were taken at different time points and analyzed for monocyte-derived cytokines by ELISA. Owing to significant donor-dependent variations in absolute cytokine levels, one representative experiment is shown in Fig. 3(AC)
. Cross-linking of CD137 by immobilized specific antibodies induced IL-8 protein in peripheral monocytes 3.8-fold (mean of four experiments) at the initial phase of culture after 2.5 h (Fig. 3A
). The overall expression of IL-8 increased with time in culture, possibly due to non-specific activation induced by adherence. Only a slight up-regulation of IL-8 (1.3-fold, mean of five experiments) by stimulation of CD137 was still observed after 6 h. Interestingly, after culture for 20 h, antibodies to CD137 markedly reduced IL-8 expression to 58%, compared to spontaneous release (mean of five experiments, Fig. 3A
).

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Fig. 3. Influence of CD137 cross-linking on the release of cytokines by monocytes. PBMC or purified monocytes were cultured in plastic culture dishes with immobilized antibody 4B4-1 to CD137 or isotype control antibodies (5 µg/ml in PBS each). Flow cytometry and immunohistochemistry, done in parallel, demonstrated monocytes as the only cell population expressing CD137. At the time points indicated, supernatants were analyzed for cytokine expression by ELISA. (A) anti-CD137 induces IL-8 in cultures of PBMC and purified monocytes. (B) Antibodies to CD137 increased TNF- production in monocytes. (C) The expression of IL-10 is decreased by anti-CD137 in both cultures of PBMC and purified monocytes. Experiments were twice performed independently and with three different healthy donors.
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Stimulation of cells with antibodies to CD137 also resulted in marked up-regulation of TNF-
protein, compared to control antibodies (Fig. 3B
). After 2.5 h of culture, TNF-
was induced 2.1-fold (mean of four experiments). A similar increase of 1.6-fold in TNF-
protein was also found after 6 h of culture. Enhanced TNF-
production after anti-CD137 was maximal after overnight culture (4.3-fold, mean of five experiments).
To prove that monocytes were the source of the increased levels of TNF-
and IL-8 induced by cross-linking of CD137, these experiments were repeated using purified primary monocytes, isolated by magnetic bead depletion of non-monocytic cells. The purified monocyte fraction contained ~96% CD14+ cells, as analyzed by flow cytometry (not shown). Stimulation of purified monocytes with antibodies to CD137 also led to an induction of IL-8. However, in contrast to monocytes in cultures of PBMC, up-regulation of IL-8 protein was still seen after 20 h in all experiments (Fig. 3A
). The production of TNF-
by purified monocytes was also markedly induced at all time points (Fig. 3B
), comparable with the results obtained with monocytes in cultures of PBMC. While induction of TNF-
was still seen after overnight culture, absolute cytokine levels had decreased by then (Fig. 3B
).
In addition to the induction of IL-8 and TNF-
, levels of IL-10 in monocytes cultured overnight with anti-CD137 were markedly reduced as compared to controls. IL-10 levels were decreased to 28% for monocytes in cultures of PBMC (mean of three experiments) and reduced to 44% for purified monocytes (mean of two experiments, Fig. 3C
). Reduction of IL-10 was already detectable after 6 h of cultures.
Competitive binding of antibody M121 to CD137 results in inhibition of TNF-
induction mediated by mAb 4B4-1
To confirm a specific interaction with CD137 as the cause for the effects of the mAb 4B4-1, we tested the ability of mAb M121, another mAb to CD137, to inhibit the induction of TNF-
release after cross-linking of CD137 by 4B4-1. Plates were precoated with isotype control or 4B4-1. In parallel, isolated PBMC were pre-incubated with soluble M121 or isotype control. Cells were then kept on precoated antibodies and after 20 h of culture supernatants were analyzed for TNF-
production. Pre-incubation of cells with mAb M121 and subsequent culture on precoated 4B4-1 resulted in TNF-
levels comparable to control samples with precoated isotype control and soluble M121 (Fig. 4
). Thus, mAb M121 specifically competes for binding to CD137 and prevents interaction of 4B4-1 with CD137 on monocytes. Pre-incubation of cells with soluble isotype control antibodies, however, allowed specific activation of CD137 by mAb 4B4-1 and therefore increased production of TNF-
as demonstrated before.
Induction of B cell death by cross-linking of CD137 on monocytes
Since CD137 is reported to be involved in cell survival (12,17), we further studied viability in different cell populations of PBMC as regulated by anti-CD137. Cross-linking of CD137 with immobilized antibodies to CD137 resulted in a significant increase of B lymphocyte death (Fig. 5A
and Table 1
). Cell death was analyzed by PI exclusion in CD19+ cells on flow cytometry. After stimulation by CD137, it was detectable in up to 25% of B lymphocytes. Time course analysis of B cell death as induced by CD137 antibodies revealed maximal induction after 48 h of culture (Fig. 5B
). Demonstration of reduced size and increased granularity of cells, another characteristic feature of cell death (31), by forward and side scatter (FSC/SSC) on flow cytometry confirmed the induction of B cell death by anti-CD137 (not shown). In view of the low absolute numbers of dead CD4 and CD8 positive T cells (Table 1
), we focused our further studies on B cell death although the rate of increase was similar in T and B cells.

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Fig. 5. Antibodies to CD137 induce cell death of B lymphocytes. PBMC from healthy donors were isolated and cultured on surfaces with immobilized antibody 4B4-1 or isotype control (5 µg/ml in PBS). Cells were then stained with antibodies to CD19 and analyzed for the exclusion of PI in flow cytometry. (A) Histograms demonstrate uptake of PI after 48 h of culture in cells gated for CD19 expression (percentage of PI+ cells). One representative experiment out of four is shown. (B) Time course analysis of PI uptake by B cells cultured with antibodies to CD137 or isotype controls. The increase in the amount of dead B cells is maximal at 48 h as compared to isotype control antibodies. Data are shown as mean ± SD of three experiments.
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Induction of B cell death by anti-CD137 is dependent on the presence of monocytes
Since monocytes had been demonstrated as the only cells expressing CD137, the induction of B cell death by antibodies to CD137 was most probably due to an interaction of B cells with monocytes. To confirm this hypothesis, cultures of cells with and without monocytes were studied. PBMC were completely depleted of monocytes, as demonstrated on flow cytometry. PBMC or monocyte-depleted cells were added to plates with immobilized antibodies and analyzed for PI uptake in B cells. Stimulation of cells by CD137 resulted in induction of B cell death only in cultures of PBMC and not in cultures of monocyte-depleted blood cells (Fig. 6
). Depletion of monocytes led to abrogation of increased cell death by anti-CD137, confirming a specific interaction with monocytes as the most likely cause.

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Fig. 6. Induction of B cell death is due to monocytes present in culture. PBMC, either untreated or completely depleted of monocytes by CD14 magnetic beads, were cultured with immobilized antibodies, as described in Fig. 4 . After 48 h, cells were stained with antibodies to CD19 and B lymphocytes were analyzed for PI exclusion by flow cytometry. Values are shown as mean ± SD percentage of PI+ B cells (n = 4). Asterisks indicate statistic significance compared to isotype control antibody (P < 0.05, by paired Student's t-test).
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In addition, co-cultures of purified monocytes and B lymphocytes isolated from peripheral blood of the same donors showed increased cell death of B lymphocytes when incubated with anti-CD137 (Fig. 7
). This indicated that the presence of monocytes and B cells was sufficient for the induction of cell death and that cross-linking of CD137 on monocytes may result in a direct interaction with B cells.

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Fig. 7. CD137-mediated cell death of B lymphocytes is due to apoptosis. Purified B cells (1x106 cells), obtained by CD19 antibodies coupled to magnetic beads and isolated monocytes (3x106), were cultured with immobilized antibodies, as described in Fig 4 . After 20 h of culture cells were fixed, permeabilized and incubated for in situ labeling of apoptosis-induced DNA strand breaks (TUNEL). After staining for CD19, the incorporation of FITC-labeled nucleotides in B cells was analyzed by flow cytometry. Histograms show labeling of DNA strand breaks in cells gated for CD19 expression. The analysis of CD137-induced apoptosis was performed twice with identical results.
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CD137-induced cell death of B lymphocytes is due to apoptosis
In order to further characterize CD137-mediated cell death, isolated B lymphocytes and monocytes were co-cultured with immobilized antibodies to CD137. To discriminate between apoptosis and necrosis, we performed an enzymatic in situ labeling technique of DNA strand breaks, a characteristic feature of apoptosis. Terminal deoxynucleotidyl transferase was used for the incorporation of FITC-labeled nucleotides to DNA strand breaks (TUNEL). After 20 h of culture with anti-CD137, cells were subjected to DNA strand break labeling, additionally stained for CD19 and analyzed by flow cytometry. Increased apoptosis was demonstrated in B cells cultured with antibodies to CD137, compared to isotype controls (Fig. 7
). The amount of apoptotic B cells induced by anti-CD137 was similar to levels with PI incorporation suggesting apoptosis as the most likely reason for B cell death.
CD137-mediated induction of B cell apoptosis is dependent on cellcell contact with monocytes
In the next set of experiments, we addressed the question whether induction of B cell apoptosis by cross-linking of CD137 on monocytes was caused by soluble factors or if a direct cellcell contact of monocytes and B cells was necessary. PBMC isolated form individual donors were divided into two fractions of cells: purified monocytes and PBMC completely depleted of monocytes by CD14 antibodies. These two fractions were separated by a porous membrane with a pore size of 0.4 µm which only allows passage of soluble mediators (Fig. 8A
). The use of this membrane prevented direct cellcell contact between B cells in the upper chamber and monocytes cultured on immobilized antibodies to CD137. As control, both fractions of cells were cultured together in parallel without separation by a membrane. As shown in Fig. 8(B)
, CD137-induced apoptosis of B lymphocytes was not seen when monocytes and B cells were separated by a membrane. Culture of isolated cell fractions without separation by a membrane showed a CD137-mediated increase in B cell apoptosis (Fig. 8B
) similar to that demonstrated before (Figs 6 and 7
).

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Fig. 8. CD137-mediated induction of B cell apoptosis by monocytes is dependent on cellcell contact. Purified monocytes were co-cultured with PBMC depleted of monocytes, separated or not separated by a porous membrane. The membrane has of a pore size of 0.4 µm which allows exchange of soluble mediators but prevents direct cellular contact between both cell fractions. (A) Sketch of experimental set-up of a co-culture separated by the membrane. (B) Cells were cultured on immobilized antibodies for 48 h and analyzed for dead B cells as described in Fig. 4 . Values are given as mean ± SD percentage of PI+ B cells (n = 4). *P < 0.05 compared to isotype control, by paired Student's t-test.
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Discussion
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This study reports on the expression of CD137 on human peripheral blood monocytes. Activation of monocytes via CD137 resulted in modulation of their cytokine expression and in increased monocyte-dependent apoptosis of B lymphocytes.
Expression of CD137 is found on various cell types and is dependent on cell activation (12,14,15). This study demonstrated constitutive expression of CD137 on monocytes in 10 out of 18 healthy donors which was strongly increased with time in culture. Under these conditions, up-regulation of CD137 protein by proinflammatory stimuli, as demonstrated by Schwarz et al. before for CD137 mRNA with phorbol esters and IL-1 (14), was not seen. This suggests maximal protein induction by contact-mediated mechanisms, as described for other monocyte genes (32).
To functionally characterize CD137 in monocytes, we first examined the expression of the monocyte-derived cytokines TNF-
, IL-8 and IL-10 in cultures of PBMC and of purified monocytes. TNF-
acts as a mediator of inflammation and is cytotoxic for many transformed cells (33). Monocytes and macrophages are the main source of TNF-
(34). IL-8, which acts as a chemoattractant for neutrophils and is involved in inflammatory reactions, is also mainly produced by monocytes (35). In our study, we found strong induction of TNF-
protein by antibodies to CD137 not only in PBMC cultures containing monocytes as the only cell population expressing CD137, but also in cultures of purified monocytes. The levels of TNF-
observed after cross-linking of CD137 on monocytes were similar to the concentrations of TNF-
reported for monocytes after CD40 stimulation (36). In parallel, mRNA transcripts for TNF-
were up-regulated in purified monocytes by signaling through CD137. The same stimulatory effect of CD137 cross-linking was also seen for IL-8 mRNA and protein, which were both markedly induced. However, in contrast to TNF-
, release of IL-8 was significantly inhibited in overnight cultures of PBMC by antibodies to CD137. Since this effect was not observed in cultures of purified monocytes, other cells such as T or B lymphocytes present in the cultures and their interaction with monocytes could interfere with CD137 signaling and the synthesis of IL-8. Levels of IL-8 induced by CD137 reached ~25% of those seen after stimulation with LPS, one of the strongest stimuli known for monocytes.
The increased production of IL-8 and TNF-
by monocytes after cross-linking of CD137 was accompanied by a marked reduction in IL-10 production in cultures of PBMC as well as in purified monocytes. IL-10 is well known as an important deactivating factor of monocyte function, e.g. by inhibiting expression of cytokines or by reducing expression of MHC class II molecules (37,38).
Taken together, these data demonstrating an increase of TNF-
and IL-8 and an inhibition of IL-10 production by monocytes argue for an association of CD137 with the proinflammatory program of monocytes.
In addition to the changes observed regarding the release of monocyte-derived cytokines, analysis of cell phenotypes in cultures of PBMC stimulated with anti-CD137 antibodies revealed an increased rate of cell death in both T and B lymphocytes. A high rate of ~10% in spontaneous B cell death has already been reported by others (39,40). However, since activation of CD137 drives up to 25% of B cells into apoptosis, this mechanism seems to be of biological relevance.
Our findings further suggest that the increased B cell death by CD137 stimulation is dependent on monocytes present in the culture. It is not mediated by soluble factors and requires direct cellcell contact. Monocyte-dependent cell death of lymphocytes has been reported by several other groups, e.g. the induction of T cell death by phorbol myristate acetate treatment of monocytes (41,42) or after cross-linking of CD4 (39). The latter mechanism is thought to be responsible for the accelerated apoptosis of T cells described in HIV infection (43). The differentiation of monocytes to macrophages in vitro by factors such as serum and the adherence of cells has been described (44,45). However, after only up to 24 h of culture, the monocytes/macrophages present in our cultures may retain many characteristics of peripheral monocytes, since complete differentiation into macrophages by adherence to plastic surfaces is found mostly after several more days of culture.
Cross-linking of CD4 on monocytes leads to death of T and B lymphocytes which has been described as being due to apoptosis mediated by the FasFas ligand pathway (39). Monocyte-dependent apoptosis of lymphocytes after activation by phorbol esters, however, does not seem to be mediated by this pathway (42). Recent experiments suggest that the apoptosis of B cells induced by cross-linking of CD137 on monocytes is also not caused by a FasFas ligand interaction. Using CD4-cross-linking as a positive control for Fas-mediated apoptosis (39), monocyte-dependent apoptosis of B lymphocytes induced by CD137 stimulation on monocytes was not inhibited by the antibody Nok-1 (not shown). This mAb is known to efficiently block interaction of Fas with its ligand (39,46).
Since a direct cellcell contact between monocytes and B cells was necessary for CD137-mediated apoptosis, other cell surface molecules than Fas also containing a `death domain' may be responsible for the apoptosis of B lymphocytes seen in our experiments. The death receptors DR3, DR4, DR5 and DR6, also members of the TNF receptor family, are potential candidates. All four are reported to induce apoptosis in lymphocytes (68, 10). In addition, transmembrane expression of TNF-
could account for apoptosis via TNF receptor I in a cellcell contact-dependent manner (47,48). Whether the up-regulation of monocyte cytokines may increase susceptibility of B cells for apoptosis, as described for CD4-mediated release of TNF-
and IFN-
by monocytes (49), remains to be determined. Functional specificity of the mAb 4B4-1, used for cross-linking of CD137 in this study, has been established earlier (50) and in our study was confirmed by competitive inhibition of 4B4-1 effects by another mAb to CD137.
Little is known about potential human ligands of CD137. The available information is almost exclusively restricted to data obtained from cell lines. Only one report which described the expression of CD137 ligand in different primary cells has demonstrated primary B cells to preferentially express CD137 ligand (51). Recently, interaction of an immobilized CD137Ig fusion protein with monocytes was reported which resulted in changes of cytokine release by monocytes. This suggested the presence of a CD137 ligand on these cells. However, expression of the CD137 ligand itself on primary monocytes was not demonstrated (52). Sequence analysis of the human CD137 ligand (12) has revealed only 36% homology to the mouse CD137 (4-1BB) ligand, compared to 7080% of homology between human and murine ligands reported for other members of the TNF family (9). In addition, there is almost no homology of the reported CD137 ligand to other members of the human TNF cytokine family (12). Therefore, other ligands with more characteristic features may exist. This assumption may be supported by recent experiments using the human kidney epithelial carcinoma cell line 293, highly efficiently transfected with cDNA of the reported human CD137 ligand. No effect of the transfected cells on the production of IL-8 by monocytes or on CD3-mediated T lymphocyte proliferation could be demonstrated (not shown).
In summary, our data suggest that signaling through CD137 activates monocytes and leads to the induction of a proinflammatory cytokine response by these cells. In addition, cross-linking of CD137 results in induction of lymphocyte apoptosis, particularly of B lymphocytes. Apoptosis of B lymphocytes is mediated by monocytes and is dependent on direct cellcell contact between monocytes and B lymphocytes.
Stimulation of monocytes by anti-CD137 and subsequent synthesis of cytokines may render lymphocytes more susceptible to cell death. Furthermore, CD137-activated monocytes could induce or increase the expression of one or more cell surface molecules causing B lymphocyte apoptosis. Interfering with the interaction of CD137 with its ligand may be a future option for modulating inflammatory reactions as well as apoptosis of B lymphocytes.
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Acknowledgments
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The support of I. Melchers and H. Eibel from the Clinical Research Unit for Rheumatology, who also provided the laboratory space, is highly appreciated. We thank S. Gross for excellent technical help, H. Illges for his help with the purification of monocytes, and A. Dinkel, K. Warnatz and H. Eibel for many interesting discussions. The plasmid pQA1 was kindly provided by D. Shire, Sanofi Research, France. This study would not have been possible without the help and encouragement of H. H. Peter.
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Abbreviations
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ILA induced by lymphocyte activation |
PBMC peripheral blood mononuclear cells |
PI propidium iodide |
TNF tumor necrosis factor |
TUNEL terminal deoxynucleotidyl transferase dUTP nick end-labeling |
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Notes
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Transmitting editor: K. Eichmann,
Received 21 July 1999,
accepted 5 October 1999.
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