290 J. Michel et al. Eur. J. Immunol. 1998.

28: 290–295

A soluble form of CD137 (ILA/4-1BB), a member of

the TNF receptor family, is released by activated
lymphocytes and is detectable in sera of patients
with rheumatoid arthritis
Jan Michel, Joachim Langstein, Ferdinand Hofstädter and Herbert Schwarz

Department of Pathology, University of Regensburg, Regensburg, Germany

CD137 (ILA/4-1BB) is a member of the tumor necrosis factor receptor family and regulates
activation, proliferation and programmed cell death in T lymphocytes. Here we show the
existence of a soluble form of CD137 (sCD137) of 16 kDa. sCD137 is released by activated
lymphocytes, and in contrast to membrane-bound CD137, expression of sCD137 seems to
be restricted to lymphocytes. sCD137 is generated by alternative splicing and two splice
variants were identified. sCD137 is present at low levels in sera of some healthy donors
(5/12; mean = 0.18 ng/ml) and is significantly enhanced in sera of patients with rheumatoid
arthritis (12/12; mean = 3.58 ng/ml).
Received 5/9/97
Revised 14/10/97
Accepted 23/10/97
Key words: Rheumatoid arthritis / Inflammation / Cytokine receptor / Human

1 Introduction Bidirectional transduction of signals exists for the CD137

receptor/ligand system. While cross-linking of CD137
The tumor necrosis factor (TNF) receptor (TNFR) family activates T lymphocytes [6, 10, 11], cross-linking of the
consists of 13 members characterized by cysteine-rich CD137 ligand has the opposite effect. This reverse sig-
pseudorepeats in their extracellular domain. TNFR I and naling through the CD137 ligand inhibits proliferation of T
II, TNFR-RP, p75 nerve growth factor receptor, CD27, lymphocytes and induces programmed cell death [10].
CD30, CD40, CD95 (Fas/Apo-1), CD137 (ILA/4-IBB),
OX40, HVEM, APO-2 (DR-4), APO-3 (DR3/WSL-1) and Soluble forms of members of the TNF receptor family
some viral homologues regulate diverse physiological have been demonstrated for the TNF and NGF recep-
programs such as proliferation, differentiation and pro- tors, CD27, CD30 and CD95 [12–16]. These soluble
grammed cell death [1–3]. receptor forms are generated by proteolytic cleavage
or alternative splicing. Soluble CD95 antagonizes
CD137 was identified in screens for receptors expressed membrane-bound CD95 and blocks CD95 ligand-
on activated lymphocytes, and originally named induced mediated cell death [15, 17]. An inhibition of the bio-
by lymphocyte activation (ILA) in man, and 4-1BB in the logical activities of TNF has also been shown for soluble
mouse [4–6]. ILA and 4-1BB recently received the HLDA TNF receptor [18]. Recombinant soluble TNF receptors
nomenclature CD137 [7]. are able to prevent septic shock and to ameliorate arthri-
tis in animal models and are currently being evaluated for
CD137 is expressed by activated T and B lymphocytes use as human therapeutics [19, 20]. Soluble TNF recep-
and monocytes and expression in primary cells is strictly tors are also employed by viruses for escaping host
activation dependent [8]. The gene for human CD137 immune responses [2].
resides on chromosome 1p36, in a cluster of related
genes, and this chromosomal region is associated with Soluble TNF receptor family members may be of diag-
mutations in several malignancies [9]. nostic value. Soluble CD30 is found in sera of patients
with CD30-positive neoplasms [14]. Soluble CD30 is also
[I 17518] present in sera of HIV-infected persons and increased
levels correlate with a fast progression to AIDS [21].
Abbreviations: mCD137: Membrane-bound CD137
sCD137: Soluble cD137

0014-2980/98/0101-290$17.50 + .50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998

Eur. J. Immunol. 1998. 28: 290–295 Soluble CD137 in RA patients 291

In the present study we demonstrate existence of Table 1. Expression of sCD137a)

soluble CD137 protein (sCD137) and show its expression
and mode of generation. Analysis of sera revealed
sCD137 ng/ml
enhanced concentrations of sCD137 in sera of patients
with rheumatoid arthritis. Control 0
PHA (10 ? g/ml); 0.5
PHA (40 ? g/ml); 3.2
PMA (1 ng/ml) + A23187 (100 nM) 0.75
2 Results PMA (2 ng/ml) + A23187 (200 nM) 2.3

a) PBMC (5 × 106) were cultured in 1 ml medium under con-

2.1 Establishment of an ELISA for soluble CD137 ditions indicated. Concentrations of sCD137 in super-
natants were determined after 3 days by ELISA.
Testing combinations of several monoclonal anti-CD137
antibodies and polyclonal antisera as capture and detec-
tion antibodies we were able to establish a sandwich
ELISA for sCD137. A fusion protein consisting of the 2.2 Expression of sCD137
extracellular domain of CD137 and the constant domain
of immunoglobulin G (CD137-Fc) served as positive con- sCD137 could be detected in supernatants of PBMC
trol and standard (Fig. 1). With this ELISA, 150 pg/ml of activated with PHA or PMA + calcium ionophore (Tab-
sCD137 can be reliably detected. The specificity of this le 1). No sCD137 was found in supernatants of resting
ELISA was confirmed by its ability to detect two other PBMC. From the supernatant of activated, but not
recombinant CD137 proteins, a histidine-tagged CD137 resting PBMC, a 16-kDa protein was immunoprecipit-
protein and a fusion protein consisting of the extracellu- ated and was detected by Western blot analysis with an
lar domain of CD137 and glutathione S-transferase anti-CD137 antibody (Fig. 2). The minor signal above the
(GST). GST, Fc and other control proteins did not result in 16-kDa band probably represents the light chain of the
a signal (not shown). precipitating antibody. Almost complete depletion of
sCD137 from supernatants after immunoprecipitation
was confirmed by ELISA (not shown).

Supernatants of the T and B lymphocyte cell lines MOLT-

4 and Raji, activated with PHA (10 and 40 ? g/ml) for 2
and 7 days were negative for sCD137 (not shown). No
sCD137 could be detected in supernatants of primary
monocytes activated with IL-1 (10 ng/ml), GM-CSF
(0.5 ng/ml) or PMA (50 ng/ml) for 1 or 3 days. Supernat-
ants of the monocytic cell lines THP-1, HL60 and U937
were negative for sCD137 (not shown). THP-1 cells acti-
vated for 2, 4 and 7 days with IL-1 (10 ng/ml) and PMA
(25 ng/ml) also did not produce sCD137 (not shown).

2.3 Generation of sCD137

Soluble receptors are generated by proteolytic cleavage

or alternative splicing. Splice variants of CD137 were
identified by RT-PCR on RNA from activated PBMC, the
supernatant of which was positive for sCD137 by ELISA.
Using primers specific for the CD137 cDNA, we obtained
two PCR products, migrating below the PCR product
derived from the full-length CD137 cDNA (not shown).
Figure 1. Specificity and sensitivity of the sCD137 ELISA.
The specificity of the ELISA was controlled by parallel pre-
coating of microtiter plates with anti-CD137 (squares) and Nucleotide sequencing identified them as splice variants
an isotype control antibody (circles). The sensitivity of the of the full-length CD137 mRNA. The larger one (sCD137-
ELISA was evaluated by serial dilutions of a recombinant 1) lacks the region from nucleotide 414 to 545, which
CD137-Fc fusion protein. encodes the serine/threonine/proline-rich segment of
292 J. Michel et al. Eur. J. Immunol. 1998. 28: 290–295

CD137. This deletion results in a frame shift, and 96 nuc-

leotides downstream in a translational stop codon.
Therefore, CD137-1 lacks the transmembrane domain
(Fig. 3), although the exon encoding the transmembrane
domain is not deleted. Sequencing of genomic DNA
identified the boundaries of the deletion as exon/intron
junctions.

In the case of the smaller PCR product (sCD137-2) the

deletion starts at the same nucleotide as in sCD137-1, at
nucleotide 414, but extends to nucleotide 681, and com-
prises the exon for the transmembrane domain and the
5' upstream exon. The 3' end of the deletion in sCD137-2
coincides with an exon/intron boundary too and the
Figure 2. Immunoprecipitation of sCD137. Supernatants of deletion also causes a frame shift with the consequence
1.5 × 108 PBMC, activated with PHA (40 ? g/ml) for 3 days, of a translational termination 15 codons downstream. No
were harvested and cellular debris was removed by centrifu- known sequence motif could be detected in the peptides
gation. Preclearing was performed with protein-G agarose, newly generated by the two frame shifts.
before immunoprecipitation with anti-CD137.

Figure 3. sCD137 is generated by differential splicing. The CD137 splice variants are schematically represented. Aligned to the
full-length CD137 cDNA (transmembrane form) are the two splice variants sCD137-1 and sCD137-2, which have deletions from
nucleotide 414 to nucleotides 545 and 681, respectively. Deletions boundaries are also indicated at the nucleotide level. The
sizes of the open reading frames of the two splice variants are indicated in amino acids (aa).
Eur. J. Immunol. 1998. 28: 290–295 Soluble CD137 in RA patients 293

Soluble CD137 is produced by activated PBMC and

purified primary lymphocytes. Surprisingly, no sCD137
was produced by monocytes, even though activated
monocytes do express mCD137 [8]. Also, no sCD137
could be detected in supernatants of several other mye-
loid and non-myeloid cell types under various activation
conditions. Production of sCD137 therefore seems to be
specific for activated lymphocytes.

Except for CD95, soluble forms of the TNF receptor

family members are generated by proteolytic cleavage.
Soluble CD95 receptors arise from differential splicing.
One splice form lacks the exon encoding the transmem-
brane domain and no frame shift is caused by this dele-
tion, a case equivalent to the murine CD137 [15]. Two
other CD95 splice products have frame shifts upstream
of the transmembrane domain resulting in truncated,
soluble CD95 proteins [17], a mechanism utilized for the
generation of human sCD137.
Figure 4. Sera of patients with rheumatoid arthritis contain
enhanced levels of sCD137. Concentration of sCD137 in Though two RNA splice variants coding for sCD137
sera of healthy donors and sera of patients with rheumatoid exist, only one sCD137 protein could be detected. Its
arthritis (RA) were determined by ELISA. Of each serum size of 16 kDa is too small to match the sCD137-1 mRNA
200 ? l were used for duplicate determinations. The horizon- with its open reading frame of 169 amino acids. The
tal bars indicate the mean values of each group. open reading frame of the shorter sCD137-2 mRNA
encodes 152 amino acids, a protein more in line with the
2.4 Soluble CD137 is enhanced in sera patients observed size of 16 kDa.
with rheumatoid arthritis
An interesting topic of future investigations will be
Having demonstrated expression of sCD137 by acti- whether sCD137 can interfere with the activities of
vated lymphocytes in vitro, we investigated whether mCD137. Cross-linking of mCD137 causes two- to four-
soluble forms of CD137 are also generated in vivo under fold enhancement of lymphocyte proliferation and is a
conditions of lymphocyte activation. Sera from healthy mechanism for B cell-mediated T cell activation.
donors were used as controls. Seven out of twelve were Engagement of mCD137 can induce an immune
negative and five contained low levels of sCD137 with a response against established tumors in mice, leading to
mean of 0.18 ng/ml for the total control group (Fig. 4). In a rejection of the tumors [11]. Production of sCD137 by
all of the twelve sera from patients with rheumatoid arth- tumors could provide a mechanism to suppress anti-
ritis, sCD137 could be detected (Fig. 4). Concentrations tumor immune responses and to evade elimination.
of sCD137 ranged from 1 to 11 ng/ml with a mean of sCD137 may also be useful in clinical situations where a
3.58 ng/ml. suppression of immune responses is desired, e.g. after
transplantations or in acute or chronic inflammatory dis-
eases. In fact, the elevated levels of sCD137 observed in
3 Discussion sera of patients with rheumatoid arthritis may reflect a
negative feed-back control of the ongoing inflammation.
Cytokines are crucial regulators of immune reactions and
their activities have to be tightly controlled to achieve a Seven of the twelve healthy donors were negative for
proper balance between an effective immune surveil- sCD137 and five contained low levels. The fact that pa-
lance and the avoidance of autoimmunity. tients with rheumatoid arthritis displayed significantly
higher concentrations of sCD137 is consistent with the
Soluble forms of receptors have been shown to seques- dependence of CD137 expression on cell activation and
ter their corresponding ligands and to antagonize the the presence of sCD137 in supernatants of activated but
activities of the membrane-bound receptors [22]. In the not resting PBMC. Almost identical to sCD137, soluble
TNF receptor family, soluble receptor forms have been NGF receptors were detected in sera of arthritis patients
identified for TNFR, NGFR, CD27, CD30 and CD95 and levels ranged from 1 to 12 ng/ml whereas no soluble
[12–16]. NGF receptors were found in sera of healthy donors [23].
294 J. Michel et al. Eur. J. Immunol. 1998. 28: 290–295

Soluble CD95 was found in sera of healthy donors and in precipitation was carried out overnight at 4 °C. Precipitates
sera of rheumatoid arthritis patients, with levels slightly were washed five times with DA100.
enhanced in the latter group (70 and 100 ng/ml, respec-
tively), [15].
4.4 Western blotting
Future research will have to elucidate whether sCD137
can be utilized as a prognostic or diagnostic factor for Precipitates were separated on a 15 % SDS polyacrylamide
specific diseases. It should also be worthwhile to exam- gel under reducing conditions and blotted onto a nitrocellu-
ine potential correlations of sCD137 with rheuma factor lose membrane (Amersham, Little Chalfont, GB). The mem-
and other clinical parameters characterizing rheumatoid brane was blocked for 1 h in APT (0.1 M Tris pH 7.4, 0.1 M
arthritis, and to test the presence of sCD137 in sera of NaCl, 2.5 mM MgCl2, 0.05 % Tween 20) 10 % dry milk,
patients with other inflammatory diseases. washed two times with APT and hybridized with chicken
anti-CD137 (7 ? g/ml) and biotinylated anti-chicken (1 ? g/ml;
Sigma), incubated in APT for 1 h at RT, washed four times in
APT and incubated with streptavidin-horseradish peroxi-
4 Materials and methods dase (1 : 1000; Dianova, Hamburg, Germany) for an addi-
tional hour. After four washes with APT for 5 min at RT, sig-
4.1 Cells and cell culture nals were visualized using the ECL system (Amersham).

Human PBMC were prepared by Ficoll gradient density cen-

trifugation as previously described [24]. Primary monocytes 4.5 Reagents
were isolated by elutriation [25]. The cell lines MOLT-4, Raji,
THP-1, HL60 and U937 were obtained from ATCC, (Rock- PHA, PMA and the calcium ionophore A23187 were pur-
ville, MD). chased from Sigma.

4.2 ELISA Acknkowledgements: We thank G. Krause, S. Rottsahl

and S. Fertig for excellent technical work. Drs. M. Widmer
Microtiter plates (Falcon 3912, Micro Test III, Becton and R. G. Goodwin (Immunex Corp., Seattle, WA) for
Dickinson, Mountain View, CA) were coated overnight at CD137-Fc protein. Sera tested were generously provided by
4 °C with anti-CD137 (1 ? g/ml, Ancell Corp., Bayport, MN) or Dr. B. Lang, University of Regensburg, Germany. This pro-
MOPC21 (Sigma, Deisenhofen, Germany) as an isotype ject was supported by the Deutsche Forschungsgemein-
control antibody. Unspecific binding was blocked by incuba- schaft.
tion with 3 % dried milk in PBS for 1 h at RT. Samples were
added for 2 h at RT and captured sCD137 was detected by
biotinylated chicken anti-CD137 antibodies (1 ? g/ml) for 1 h
at RT, followed by incubation with streptavidin-alkaline 5 References
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