Identification and characterization of a novel spliced variant that encodes human soluble tumor necrosis factor receptor 2
Begoña Lainez1,2,
José Manuel Fernandez-Real2,
Xavier Romero1,
Enric Esplugues3,
Juan D. Cañete4,
Wifredo Ricart2 and
Pablo Engel1
1 Immunology Unit, Department of Cellular Biology and Pathology, Medical School, University of Barcelona, Institut dInvestigacions Biomèdiques August Pi i Sunyer, 08036 Barcelona, Spain 2 Diabetes Unit, Endocrinology and Nutrition, University Hospital of Girona Dr Josep Trueta, 17007 Girona, Spain 3 Department of Physiology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain 4 Department of Rheumatology, Hospital Clinic, Barcelona, Institut dInvestigacions Biomèdiques August Pi i Sunyer, 08036 Barcelona, Spain
Correspondence to: P. Engel; E-mail: engel{at}medicina.ub.es
Transmitting editor: D. Wallach
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Abstract
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Tumor necrosis factor (TNF)-
is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation, differentiation and cell death induction in several cell types. The biological effects of TNF-
are mediated via the cell-surface TNF receptors TNFR1 and TNFR2. Soluble forms of these two receptors, which contain the extracellular ectodomains, are proteolytically cleaved from the membrane. High levels of soluble (s) TNFR2 in serum have been documented in multiple inflammatory pathologies. We describe here a new differential spliced isoform of human TNFR2 missing exons 7 and 8, DS-TNFR2(
7,8). This novel isoform lacks the transmembrane and cytoplasmic domains. Expression studies with DS-TNFR2(
7,8) cDNA transiently transfected COS cells showed that it encodes a sTNFR2 receptor of
42 kDa. Soluble DS-TNFR2(
7,8) blocked TNF-
-induced apoptosis, which suggests that it regulates TNF-
function by antagonizing its biological activity. An ELISA was developed that quantifies sTNFR2 generated by alternative splicing. Our data show that sTNFR2 generated by alternative splicing can be found in sera of healthy individuals, at increased levels in patients with sepsis and at high concentrations in rheumatoid arthritis patients.
Keywords: inflammation, soluble cytokine receptor, tumor necrosis factor receptor
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Introduction
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Tumor necrosis factor (TNF)-
is a potent cytokine that plays a pivotal role in immune responses to infection by eliciting a wide spectrum of cellular responses, including induction of adhesion molecules and other cytokines, cell proliferation, differentiation, and apoptosis (1,2). Many cell types produce TNF-
, including monocytes/macrophages, lymphocytes, keratinocytes and fibroblasts, in response to inflammation, infection, tissue injury or other environmental challenges. The principal physiologic function of TNF-
is to stimulate the recruitment of leukocytes to sites of infection and to activate these cells to eliminate microorganisms (3). However, excessive TNF-
production is responsible for many of the systemic complications of severe infections. In fact, acute release of large amounts of TNF-
into the bloodstream is followed by the characteristic manifestations of septic shock (4).
TNF-
exerts its effects through two distinct receptors, TNFR type I (TNFR1; TNF-R55; CD120a) and TNFR type II (TNFR2; TNFR75; CD120b) (5,6). Both belong to the TNFR superfamily and bind TNF-
with high affinity (7). TNFR1 is expressed constitutively on a broad spectrum of cell types. In contrast, the expression of TNFR2 is restricted to endothelial cells and cells of hematopoietic origin, and modulated by a variety of stimuli (8). Binding of TNF-
to TNFR1 and TNFR2 induces the recruitment of several signaling molecules to the cytoplasmic domains of these receptors that activate various signaling cascades leading to the activation of caspases, and transcription factors AP-1 and NF-
B (9,10). The existence of a similar extracellular domain, characterized by cysteine-rich pseudo-repeats, but dissimilar intracellular domains, has led to the suggestion that these two receptors transduce distinct signals (10). Soluble receptors are part of an expanding group of regulatory molecules that are derived from the extracellular domains of integral cell-surface receptors (11). These include soluble adhesion molecules, and receptors of cytokines and growth factors. Their ligand-binding affinities are often similar to those of cell-surface receptors, suggesting that their biological role is to modulate the action of their ligands. Two independent cellular processes responsible for the generation of these soluble receptors have been identified (12). First, differential mRNA splicing can lead to soluble factors that lack the membrane-spanning domains of the cell-associated protein [receptors for IL-4, granulocyte macrophage colony stimulating factor (GM-CSF), LIFR and IL-11]. The second mechanism involves the proteolytic cleavage of the membrane-anchored protein at a site proximal to the cell surface (CD62 ligand, transforming growth factor-ß receptor and platelet-derived growth factor receptor) (13).
Proteolytic cleavage is suggested as the mechanism of generation of soluble (s) TNFR. Cellular activation by agents such as lipopolysaccharide, anti-CD3 antibodies, fMLP, GM-SCF, calcium ionophore, phorbol 12-myristate 13-acetate (PMA) and its natural ligand TNF induced rapid shedding of membrane TNFR in several cell types, including monocytes/macrophages, T lymphocytes and granulocytes (1417). sTNF-
receptors that maintain their ability to bind to TNF-
with high affinity limit the bioavailability of TNF-
by competing for the cytokine with the cellular receptor (18,19). sTNFR are present constitutively in serum at concentrations that increase significantly in infectious diseases, cancer patients and autoimmune diseases. Increased concentrations of sTNFR were first detected in patients with cancer (20). High levels have been also detected in a variety of hematopoietic malignancies (21). In HIV infection and sepsis, sTNFR2 concentrations strongly correlate with the clinical stage and the progression of the disease, and can be of predictive value (22,23). High levels of TNFR2 have been found in the sera of patients with several autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, progressive systemic sclerosis and mixed connective disease (2427). Elevated levels have been also found in chronic pathologies such as Type 2 diabetes mellitus (24,28,29).
Here, we provide direct evidence for the existence of a biologically active form of sTNFR2 produced by differential splicing. We also demonstrate that sTNFR2 generated by alternative splicing is present in the serum of normal healthy individuals and at high levels in patients with rheumatoid arthritis.
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Methods
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Cells
Peripheral blood mononuclear cells were isolated using Ficoll-Hystopaque (Sigma, St Louis, MO) according to the manufacturers instructions. COS-7, U937 and L929 cells were obtained from the ATCC (Manassas, VA) and maintained in DMEM (Life Technologies, Rockville, MD), containing 10% heat-inactivated FCS (Life Technologies), 2 mmol/l L-glutamine and 100 U/ml penicillin/streptomycin (Life Technologies).
Cloning of TNFR2 cDNA
Total RNA was isolated from human peripheral blood mononuclear cells with TRIzol reagent (Life Technologies). cDNA was synthesized from 1 µg of total RNA using the First-Strand cDNA synthesis kit (Boehringer Mannheim, Mannheim, Germany). The 5' primer was 5'-GCA CCC ATG GCG CCC GTC-3' and the 3' primer was 5'-CAG CCC ACA CCG GCC TGG-3'. TNFR2 full-length and DS-TNFR2 were subcloned in pCR3.1 expression vector (Invitrogen, Carlsbad, CA). Sequencing was performed with the Big Dye terminator kit (Perkin-Elmer, Foster City, CA), according to the manufacturers instructions.
Expression of DS-sTNFR2(
7,8)
COS-7 cells were transiently transfected with Lipofectamine Plus reagent (Life Technologies) with 4 µg of empty pCR3.1 vector (negative transfection control) or with 4 µg pCR3.1 vector containing the full-length TNFR2 or DS-TNFR2 cDNA. After 24 h, transfected COS-7 cells were transferred to sterile glass coverslips in six-well culture plates (Costar, High Wycombe, UK). In some experiments, COS cells were treated with 1 µg/ml Brefeldin A (Becton Dickinson, San Jose, CA) for 4 h at 37°C, 5% CO2.
Immunocytochemistry analysis was performed using indirect immunofluorescence. Briefly, cells were washed once with PBS (2% FCS), incubated with blocking solution (3% BSA/10% FCS in PBS) for 30 min and then stained with biotinylated mouse mAb to human CD120b [clone 4D1B10 (MR2-1) mouse IgG1] (Caltag, Burlingame, CA) for 30 min at room temperature. After the cells were washed 3 times with PBS (2% FCS) for 5 min, streptavidinIndocarbocyanin Cy3 (Jackson ImmunoResearch, West Grove, PA) was added and incubated for 30 min at room temperature. The cells were then washed and fixed in methanol (20°C) for 15 min. The coverslips were mounted on slides with mounting medium and examined using a Nikon microscope Optiphot-2 (Nikon, Kingston upon Thames, UK). Photomicrographs were taken with x20 and x40 objectives. Cytoplasmic staining was carried out by pre-treatment of the cells with methanol (20°C) for 15 min to allow fixation and permeabilization, and then performed as in surface staining. Purified mouse IgG was used as a negative control.
sTNFR2 detection in supernatant of cultured cells
Two days after transient transfection of COS-7 cells with full-length TNFR2 or DS-TNFR2(
7,8) cDNA, cells were washed and incubated with medium or 50 ng/ml of PMA for 30 min and 12 h at 37°C. Peripheral blood mononuclear cells and U937 cells (5 x 106/ml) were incubated with medium or PMA (10 ng/ml) for 24 h at 37°C. Supernatants were recovered, centrifuged at 3500 r.p.m. for 10 min and stored at 80°C.
Immunoprecipitation and immunoblotting of DS-sTNFR2(
7,8)
DS-TNFR2 and full-length TNFR2 transfected COS cells were washed once in PBS buffer and lysed in 1 ml buffer containing 1% (v/v) Nonidet P-40 and protease inhibitors. Immuno precipitations were carried out using 5 µg of mAb. Cell lysates were pre-cleared once for 30 min using only Protein ASepharose beds and twice for 30 min using 40 µl (50% v/v) murine Ig-coated beds. The pre-cleared lysate was then incubated with 40 µl of mouse anti-human CD120b-coated beds or mouse Ig-coated beds with constant rotation at 4°C for 18 h. Immunoprecipitates were washed and analyzed by SDSPAGE. Samples were run in the presence of 5% ß-mercaptoethanol. The mol. wt was determined using pre-stained standard mol. wt markers (Bio-Rad, Hercules, CA). Proteins were transferred to PVDF membranes (Immobilon; Millipore, Boston, MA). After 1 h of incubation at room temperature with blocking solution (3% non-fat milk in PBS), membrane was incubated for 1 h at room temperature with rabbit anti-human CD120b (Monosan, Uden, The Netherlands) at 1 µg/ml in Tris-buffered saline with 0.2% Tween 20 (TTBS). After washing with TTBS 3 times for 5 min, horseradish peroxidase-conjugated anti-rabbit antibody (Amersham Biosciences, Little Chalfont, UK) was added for 30 min. Membrane was washed with TTBS as before and the blot was developed using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL).
TNF-
cytotoxicity assay
L929 were plated by adding 3 x 104 cells to each well of a 96-well plate and treated with 8 µg/ml of actinomycin D. Cells were incubated with different concentrations (0.1 and 1 ng/ml) of TNF-
(Sigma) in presence of supernatant derived from COS-7 cells transfected with either DS-TNFR2 cDNA or with empty vector. After 18 h in a 37°C, 5% CO2 incubator, cells were washed with PBS and stained with 0.05% Crystal Violet in 20% ethanol for 10 min at room temperature. After washing with cold water 3 times, and overnight drying, 100 µl of methanol was added and absorbance at 595 nm was measured. Percentages of cell survival values were calculated with the formula: 100 x (OD sample OD maximum lysis)/(OD medium control cell OD maximum lysis).
Production of a mAb against TNFR2
BALB/c mice were immunized 3 times, at 2-week intervals, with the 14-residue peptide (PMGPSPPAEGSTGD) conjugated to keyhole limpet hemocyanin. This synthetic peptide corresponds to the sequence of the membrane-proximal region of TNFR2 and was obtained from the Peptide Synthesis Service of the Chemical School of the University of Barcelona. The first i.p. injection consisted of 30 µg of conjugated peptide in 200 µl of PBS mix with 200 µl of complete Freunds adjuvant. The second i.p. injection consisted of 30 µg of conjugated peptide in 200 µl of PBS and 200 µl of incomplete Freunds adjuvant. The final dose consisted of 30 µg/100 µl without adjuvant injected i.v. The mice were sacrificed on the third day after the final boost. Spleen cells from immunized animals were fused with NS-1 myeloma cells (European Collection of Cell Cultures, Salisbury, UK) using polyethylene glycol solution (Sigma). Supernatant fluid from 650 hybridoma-containing wells was screened by ELISA using peptide-bound 96-well microplates (Corning-Costar, Cambridge, MA). Plates were coated with 100 µl of 4 µg/ml of peptide in PBS overnight at 4°C and blocked with 250 µl of 2% BSA in PBS at 37°C for 1 h. Aliquots of 100 µl of supernatant diluted 1:2 in PBS containing 2% BSA were incubated for 1 h at room temperature. Bound mAb were detected with a horseradish peroxidase-conjugated anti-mouse mAb (Sigma) and reaction developed by o-phenyl enediamine substrate (Sigma). Hybridomas were subcloned by limiting dilution. mAb were purified with a Protein A column (Bio-Rad) from concentrated supernatant obtained from the culture of the hybridomas in CL 350 flasks (Integra Biosciences, Chur, Switzerland). Purified mAb were dialyzed extensively against PBS, aliquoted and stored at 20°C. Antibody isotype was determined using a mouse mAb isotyping kit (Boehringer Mannheim, Mannheim, Germany).
Detection of sTNFR2 by quantitative ELISA
sTNFR2 was detected by sTNFR (80 kDa) human ELISA Module Set (Bender MedSystems, Vienna, Austria). Briefly, each well of the microplates (Corning-Costar) was coated with 100 µl of mAb directed against TNFR2, left overnight at 4°C, blocked and reacted with 100 µl of serum diluted 1:10 or 1:5 in PBS containing 2% BSA. Bound sTNFR2 protein was detected with a peroxidase-conjugated anti-human TNFR2 mAb. Reaction was developed by TMB substrate and stopped with 4N H2SO4. Absorbance was measured at 450 nm.
Detection of sTNFR2 generated by alternative splicing by quantitative ELISA (DS-sTNFR2-ELISA)
The levels of DS-sTNFR2 protein were determined using a modified version of the sTNFR (80 kDa) human ELISA Module Set (Bender MedSystems). The anti-TNFR2 mAb produced in this study (TNFR2 clone 572) was used as coating antibody (5 µg/ml). Bound sTNFR2 protein was detected with the peroxidase-conjugated anti-human TNFR2 mAb provided by the commercial kit. The same standard of the commercial kit could be use for this ELISA since it comprised the complete extracellular region of the TNFR2 (from amino acid number 1 to 235) and mAb TNFR2 clone 572 was raised against a peptide of TNFR2 spanning from amino acids number 222 to 235.
The sensitivity of the two ELISAs was very similar: 0.15 ng/ml in the commercial ELISA and 0.25 ng/ml in the ELISA developed in this study. Since the sera were diluted 1/5, the lower detection levels of the ELISAs were 0.75 and 1.25 ng/ml respectively.
Subjects
Peripheral venous blood was drawn from healthy volunteers, patients with sepsis confirmed by positive blood culture for bacteria and patients with rheumatoid arthritis.
Healthy subjects were of Caucasian origin; none had taken any medication, or had any systemic disease or infection in the previous months. We analyzed 145 serum samples from healthy volunteers (93 men and 52 women) with a mean age of 39.7 ± 11.7 years, range 1969 years. Thirty serum samples were obtained from patients with sepsis (21 men and nine women) with a mean age of 61.4 ± 23.1, range 186 years. Thirty-nine sera from patients fulfilling American College of Rheumatology criteria for rheumatoid arthritis were included in this study (30). Demographic and clinical characteristics of the patients were as follow (nine men and 30 women): age 52.2 ± 15.7 years; disease duration 9.5 ± 6.5 months; rheumatoid factor positive 78.3%; HLA-DRB1-04 50%; erosive disease 21.7%. The Hospital Dr Josep Trueta Ethics Committee and Hospital Clinic Ethics Committee approved the protocol. Informed consent was obtained from each subject and the samples were obtained according to Declaration of Helsinki principles.
Statistical analysis
The data obtained from serum samples are reported as mean ± SEM. Differences between groups were assessed by one-way ANOVA followed by post hoc analysis using Dunns or Bonferroni correction as appropriate. Statistical significance was set at P < 0.05.
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Results
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Cloning of DS-TNFR2(
7,8) cDNA
A splice variant of TNFR2 was cloned using primers to amplify full-length TNFR2 by RT-PCR from RNA from peripheral blood mononuclear cells. The sequence was submitted to the GenBank (accession no. AY148473). The ratio between clones containing the spliced variant and the full length, obtained from peripheral blood mononuclear cells, was 1:92. This short TNFR2 isoform was characterized by a deletion of 113 bp, starting at nucleotide position 788 and ending at position 900 of the full-length TNFR2 cDNA (Fig. 1A). This 113 bp corresponds to the sequence of exons 7 and 8 (Fig. 1A and B). This deletion resulted in a cDNA encoding for a putative TNFR2 protein retaining the extracellular domain, but lacking 27 of 32 amino acids of the transmembrane domain and the cytoplasmic region. Due to a frame shift, a unique sequence ASLACR in its C-terminus was generated (Fig. 1A and B). This differential spliced product is likely to encode a soluble secreted isoform of TNFR2. Thus, we called this isoform Differential Splicing sTNFR2 (DS-sTNFR2
7,8) and the soluble isoform of TNFR2 generated by Proteolytic Cleavage: PC-sTNFR2.

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Fig. 1. Sequence of TNFR2 differential splicing isoform. (A) cDNA sequence of TNFR2 differential splicing form DS-sTNFR2( 7,8) (accession no. AY148473) compared with the full-length TNFR2 (accession no. M32315). Spliced nucleotides are in bold and exons are indicated in numbers above the cDNA sequence. Exon boundaries were deduced from the TNFR2 genomic sequence (accession no. U52165). The transmembrane domain is boxed and the stop codon is indicated by an asterisk. (B) Diagram of the frame shift produced by the alternative splicing. The primers used are indicated by arrows. Positions of the first ATG and the stop codon are indicated. The amino acids produced after the frame shift are in bold.
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DS-TNFR2(
7,8) encodes a soluble receptor
To study the DS-TNFR2(
7,8) translation product, levels of sTNFR2 were measured in supernatant of COS transfected cells (Fig. 2). sTNFR2 was detected in supernatant of DS-TNFR2(
7,8) cDNA transfected cells, but not in supernatant of full-length TNFR2 cDNA transfected cells (Fig. 2). Only after treatment with the shedding inducing agent PMA were significant levels of sTNFR2 detected in the supernatant of cells transfected with full-length TNFR2 cDNA (Fig. 2). PMA did not affect the levels of sTNF in DS-TNFR2(
7,8) transfected cells. These results showed that the DS-TNFR2(
7,8) cDNA product was not retained in the membrane, as the full-length TNFR2 form was, and was directly secreted into the medium.

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Fig. 2. sTNFR2 levels detected in the supernatant of transfected cells. Supernatant of DS-sTNFR2( 7,8), TNFR2 or mock transfected COS-7 cells with PMA treatment (solid squares) or without (open squares) were tested for sTNFR2 by ELISA.
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Detection of DS-sTNFR2(
7,8) in transfected cells
Immunocytochemical analysis revealed an intense staining of both the cell-surface and cytoplasm of cells transfected with the full-length TNFR2, but no significant staining could be detected in COS cells transfected with DS-TNFR2(
7,8) (Fig. 3). However, when cells were pre-treated with the protein transport inhibitor Brefeldin A, TNFR2 protein could also be detected in DS-TNFR2(
7,8) transfected cells (Fig. 3). Moreover, DS-TNFR2(
7,8) protein product was immunoprecipitated from Brefeldin A transfected cells, showing a mol. wt of
4045 kDa (Fig. 4).

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Fig. 3. Immunocytochemistry of TNFR2 transfected cells. COS-7 cells transfected with TNFR2 (left panels) and transfected with DS-sTNFR2( 7,8) cDNA (right panels). For surface staining (top panels), transfected cells were detected with a biotinylated anti-TNFR2 mAb directed against the extracellular domain and avidin-conjugated Cy3. For intracellular staining (middle and bottom panels), cells were fixed and permeabilized before the staining. In the bottom panels, cells were pre-treated with Brefeldin A for 4 h.
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DS-sTNFR2(
7,8) blocks TNF-
biological activity
The ability of DS-sTNFR2(
7,8) to bind and neutralize human TNF-
was studied by a TNF-
-mediated cytotoxicity assay. Results indicate that supernatants from DS-TNFR2(
7,8) transfected cells, but not those from empty vector transfected cells, inhibited TNF-
-induced cell death (Fig. 5A). DS-sTNFR2(
7,8) inhibited TNF-
-induced cytotoxicity in a dose-dependent manner. Concentrations of DS-sTNFR2(
7,8) as low as 4 ng/ml were able to inhibit 100% of the cytotoxicity induced by 1 ng/ml of TNF-
(Fig. 5B).
Development of an ELISA that quantifies sTNFR2 generated by alternative splicing
The isolation of a spliced variant of TNFR2 encoding a soluble receptor prompted us to develop an ELISA able to quantify sTNFR2 generated by alternative splicing. The conventional ELISA systems that measure sTNFR2 do not differentiate between the soluble receptor generated by alternative splicing (DS-sTNFR2) or proteolytic cleavage (PC-sTNFR2). In order to specifically quantify DS-sTNFR2, we produced a mAb against an epitope present in TNFR2, but predicted to be absent in soluble PC-TNFR2 (31,32). A mAb TNFR2 clone 572 was raised by immunizing mice with a peptide that corresponds to the first 14 residues of the membrane-proximal region. This mAb was used as capture antibody in the DS-sTNFR2 ELISA as described in Methods. In order to shown that this ELISA was not detecting PC-TNFR2 we induce in vitro the shedding of TNFR2 from several cells, including TNFR2 transfected COS-7, peripheral blood mononuclear cells and the U937 monocytic cell line, using PMA. The DS-TNFR2 ELISA did not detected shed TNFR2 from any of these cells (Fig. 6A and B). Both DS-TNFR2 ELISA and conventional ELISA detected sTNFR2 in the supernatant of COS cells transfected with DS-TNFR2(
7,8) (Fig. 6C).

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Fig. 6. Specificity of DS-sTNFR2 ELISA. Full-length TNFR2 transfected COS cells (A) and different cellular types (B) were stimulated with 50 and 10 ng/ml of PMA respectively, and supernatants containing shed receptor were tested by both DS-sTNFR2 and sTNFR2 ELISA. (C) Supernatant of DS-sTNFR2( 7,8) transfected cells measured by both ELISAs.
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Detection of sTNFR2 generated by alternative splicing in sera of healthy control subjects, and patients with sepsis and rheumatoid arthritis
Low serum levels of sTNFR2 generated by alternative splicing were found in the serum of healthy control subjects (n = 145): 1.7 ± 0.3 ng/ml, range 018.5 ng/ml) (Fig. 7); 50.3% of control subjects had no detectable levels of DS-sTNFR2. Mean percentage DS-sTNFR2 out of total TNFR2 levels was 21.1 ± 2.4% and only in 18% of control subjects did DS-sTNFR2 represent >50% of the total sTNFR2.

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Fig. 7. Levels of sTNFR2 in healthy, septic and rheumatoid arthritis subjects. DS-sTNFR2 (solid squares) and sTNFR2 (open squares) levels as determined by ELISA.
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The serum levels of sTNFR2 were significantly elevated in patients with sepsis (n = 30) (35.4 ± 5.9 ng/ml, range 6.3147.5 ng/ml) compared to control subjects (P < 0.0001). These patients tended to have higher serum levels of DS-sTNFR2 than control (8.6 ± 3.0; range: 065.0); however, values did not reach statistically significant difference, P = 0.10 (Fig. 7). Patients with rheumatoid arthritis had significantly higher levels of sTNFR2 than control subjects (36.9 ± 7.9 ng/ml, range 1.8161.5 ng/ml, P < 0.001) (Fig. 7). Moreover, these patients had significantly higher serum levels of DS-sTNFR2 than control subjects (17.2 ± 5.5; range 0143. 6, n = 39, P < 0.001) (Fig. 7). These data show that sTNFR2 produced by shedding was predominant in the serum of healthy subjects and patients with sepsis. In patients with high levels of sTNFR2 (>30 ng/ml), DS-sTNFR2 represented 22.4% in the sera of sepsis and 44.8% in the sera of rheumatoid arthritis.
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Discussion
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In this study we identified and characterized a novel alternatively spliced human TNFR2 mRNA isoform. This isoform encodes a soluble protein, which we named DS-sTNFR2(
7,8), which inhibits TNF-
biological activity. Due to splicing, cDNA encoding DS-sTNFR2(
7,8) lacks exons 7 and 8. The isolated mRNA had a 113-bp deletion that gave rise to a frame shift resulting in a stop codon a few bases after the deletion. The splicing event is such that the N-terminus residues corresponding to the extracellular domain of the full-length TNFR2 remained exactly the same, followed by the first 5 amino acids of the transmembrane domain and a unique 6-amino-acid tail on DS-sTNFR2(
7,8) in its C-terminus. Deletion of most of the transmembrane domain suggested that DS-sTNFR2(
7,8) protein may not be retained as a transmembrane protein and may be soluble. Evidence of its soluble nature was the detection of sTNFR2 in the culture supernatant of COS cells transiently transfected with cDNA of DS-sTNFR2(
7,8). In contrast, COS cells transfected with the full-length TNFR2 cDNA produced significant amounts of soluble receptor only after treatment with PMA-induced shedding. Furthermore, immunocytochemistry on DS-sTNFR2(
7,8)-COS transfected cells revealed a pattern concordant with a soluble protein, with no membrane staining and weak intracytoplasmic staining that significantly increased after the treatment with the transport inhibitor Brefeldin A. Western blot analysis showed that DS-sTNFR2(
7,8) is a protein of
42 kDa, 24 kDa lower than full-length TNFR2. Our TNF-
bioassay data indicate that DS-sTNFR2(
7,8) neutralizes the activity of TNF-
, which is not surprising since the extracellular domain is maintained identical to full-length TNFR2. Thus, these data provide evidence that, besides shedding, alternative splicing produces sTNFR2. Although we have no experimental evidence, an isoform lacking only exon 7 could also encode a soluble product of this receptor. Two mouse TNFR2 mRNAs have been detected by northern blot analysis, whether these mRNAs are due to the same differential splicing shown here remains unknown (33,34).
Alternative splicing is a powerful and versatile regulatory mechanism that affects quantitative control gene expression and functional diversification of protein (35). Other members of the TNFR family (CD95 and CD137) that are found as soluble receptors are generated by alternative splicing (3638). However, alternative splicing and shedding, the two mechanisms of producing soluble transmembrane receptors, are not mutually exclusive. The soluble IL-6 receptor, IL-4 receptor, growth hormone receptors and CD40, a member of the TNFR family, are examples of receptors generated by both mechanisms (13,3941).
Here, we described the development of an ELISA that specifically recognizes sTNFR2 generated by alternative splicing. Our data show that DS-sTNFR2 was present at low concentration in the serum of healthy individuals and that this concentration represents 21% of the overall levels of sTNFR2. We analyzed the presence of sTNFR2 in two pathological conditions where elevated levels of this soluble protein have been reported (23,24). Levels of DS-sTNFR2 were higher in sera of patients with sepsis. However, DS-sTNFR2 represented only 22% of all sTNFR2, indicating that, at least in this pathological condition, shedding generates the predominant form of sTNFR2. These data are consistent with our observation that leukocytes in vitro stimulated with lipopolysaccharide produced mainly PC-sTNFR2 (data not shown). Thus, in an acute inflammatory response as seen during sepsis, most of the sTNFR2 would correspond to shed receptor. High concentrations of DS-sTNFR2 were found in the serum of patients with rheumatoid arthritis. In contrast to sepsis, in rheumatoid arthritis with high levels of sTNFR2, about half of the soluble receptor was generated by alternative splicing. Our data analysis showed no correlation between the levels of DS-TNFR2 and PC-TNFR2, suggesting the existence of two independent regulatory pathways of sTNFR2 release, possibly with different consequences for the regulation of TNF bioactivity.
The actual involvement of sTNFR2 in disease pathogenesis remains controversial. It has been suggested that it may act as an antagonist of TNF-
action by competing with cell-surface receptors, but also as an agonist by protecting TNF-
from degradation, and therefore stabilizing the TNF-
trimeric structure and prolonging TNF-
s availability for binding to membrane receptors (13). Its biological activity probably depends on the relative concentrations of ligand and soluble receptor. It has been reported that sTNFR at low concentration enhances the actions of TNF-
, but higher concentrations of sTNFR cancel out the effects of TNF (42).
Transgenic mice constitutively overexpressing 3- to 4-fold higher levels of human TNFR2 than control mice develop multi-organ inflammation (43). Although these mice express high levels of sTNFR2, the pathogenesis in this animal model may be not related to the soluble receptor, but to the overexpression of the membrane-bound receptor. In keeping with this observation, it has been suggested that increased levels of shed TNFR2, as seen in human diseases, reflect a similar up-regulation of the cell-surface form of the receptor, which may affect immune homeostasis and/or pathogenesis. Our data open the possibility that in certain pathologies an important part of the soluble receptor may not be related to the shedding of the cell-surface expressed receptor, but to the induction of DS-sTNFR2. Knowing the relative contribution of each of the two soluble isoforms to the pool of circulating soluble serum TNFR2 in patients with several inflammatory diseases may give additional insight into their biological and pathological relevance.
Increasing evidence implicates dysregulation of TNF-
expression and/or signaling in the pathology of many diseases, including rheumatoid arthritis, Crohns disease and neuropathologies, multiple sclerosis, and Alzheimers disease (44). Moreover, a human disease called TNF-receptor-associated periodic syndrome has been shown recently to be caused by dominantly inherited mutations in the gene encoding TNFR1 that affect its shedding from the cell surface (45). Perhaps more importantly, a TNFR2Fc hybrid molecule has been shown to be very useful in the treatment of patients with rheumatoid arthritis (46). This is, in fact, the first soluble cytokine receptor to receive approval as a therapeutic agent for human use. Further research will have to elucidate whether DS-sTNFR2 can be utilized as a prognostic or diagnostic factor for specific diseases. A better appreciation of how sTNF-
receptors production is regulated will be required to understand the pathophysiological role of TNF in a broad spectrum of diseases.
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Acknowledgements
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We are grateful to Merçé Alsius for collecting the sera of the sepsis patients. This study was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (SAF00-0037) and from the Fondo de Investigaciones Sanitarias (00/0024-01 and 00/0023-03). J. D. C. was supported by Aventis Pharma SA. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession no. AY148473.
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Abbreviations
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DS-sTNFR2(
7,8)soluble TNFR2 generated by differential splicing
GM-CSFgranulocyte macrophage colony stimulating factor
PC-sTNFR2soluble TNFR2 generated by proteolytic cleavage
PMAphorbol 12-myristate 13-acetate
sTNFR2soluble TNF receptor 2
TNFtumor necrosis factor
TNFR2TNF receptor 2
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