The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 4239-4248
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rossol, M.
Right arrow Articles by Wagner, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rossol, M.
Right arrow Articles by Wagner, U.

Interaction between Transmembrane TNF and TNFR1/2 Mediates the Activation of Monocytes by Contact with T Cells1

Manuela Rossol*, Undine Meusch*, Matthias Pierer*, Sylke Kaltenhäuser*, Holm Häntzschel*, Sunna Hauschildt{dagger} and Ulf Wagner2,*

* Department of Medicine IV, University of Leipzig, Leipzig, Germany; and {dagger} Department of Immunobiology, Institute of Biology II, University of Leipzig, Leipzig, Germany


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 Disclosures
 References
 
Monocytes and monocytic cells produce proinflammatory cytokines upon direct cell contact with activated T cells. In the autoimmune disease rheumatoid arthritis, the pivotal role of TNF-{alpha} implies that the interaction between transmembrane TNF-{alpha} (mTNF) and the TNF receptors (TNFR1 and TNFR2) might participate in the T cell contact-dependent activation of monocytes. Accordingly, treatment of rheumatoid arthritis by administration of a TNF-{alpha}-blocking Ab was found to significantly decrease TNF-{alpha} production by monocytes. Several lines of evidence indicated that signaling through TNFR1/2 and through mTNF (reverse signaling) is involved in TNF-{alpha} production by monocytes after T cell contact: 1) blocking mTNF on activated T cells leads to a significant reduction in TNF-{alpha} production; 2) down-regulation of TNFR1/2 on monocytes by transfection with small interfering RNA results in diminished TNF-{alpha} production; 3) blocking or down-regulating TNFR2 on activated T cells inhibits TNF-{alpha} production, indicating that mTNF on the monocyte surface mediates signaling; 4) ligation of mTNF on monocytes by surface TNFR2 transfected into resting T cells induces TNF-{alpha} production due to reverse signaling by mTNF; and 5) ligation of mTNF on monocytes by a soluble TNFR2:Ig receptor construct induces TNF-{alpha} production due to reverse signaling. In conclusion, we identified mTNF and TNFR1/2 as interaction partners contributing to TNF-{alpha} production in monocytes. Both pathways initiated by mTNF-TNFR interaction are likely to be inhibited by treatment with anti-TNF-{alpha} Abs.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 Disclosures
 References
 
In recent years, studies pioneered by the group of Dayer and colleagues and work from several other laboratories (reviewed in Refs. 1 and 2) have shown that activated T cells are potent inducers of cytokine production in monocytes and macrophages (reviewed in Refs. 1 and 2). This mode of activation, which requires direct cell-cell contact, has also been implicated in the pathogenesis of rheumatoid arthritis (RA),3 an autoimmune disease associated with the production of high levels of monocytic cytokines like IL-1beta and TNF-{alpha} in the absence of microbial stimuli. T cells have been shown to be clonally expanded in RA and to exhibit pathological phenotypes, including the expression of new surface molecules. Thus, the contact of those pathologically activated T cells with monocytes could induce cytokine production and contribute to the disease. T cells isolated from the synovial membrane of RA patients are indeed able to induce cytokine production in monocytes at levels comparable to those induced by in vitro-stimulated T cells (3, 4).

The surface molecules contributing to the T cell-dependent monocyte activation are not conclusively identified. Several ligand-receptor pairs such as CD40 and CD40 ligand have been suggested to be involved, although primarily in mature macrophages rather than in monocytes (5, 6). Furthermore, Abs against CD2, LFA-1, CD69, and ICAM-1 were all found to partially inhibit T cell-dependent monocyte activation (4, 7, 8).

In addition, another group of molecules, namely, membrane-anchored cytokines, also seem to participate in this mode of activation. Among these cytokines are IFN-{gamma} (9) and IL-1 (7, 10). In view of the fact that TNF-{alpha} plays a pivotal role in the initiation and perpetuation of RA, the role not only of its soluble, but also of its transmembrane form (mTNF) is of special interest. The T cell-dependent IL-10 production of monocytes was shown to be regulated by mTNF (11). Addition of anti-TNF-{alpha} Ab prevents TNF-{alpha} production during direct T cell contact, which can be interpreted as evidence for a role of mTNF (12), although soluble TNF-{alpha} is also likely involved in this interaction. However, steric hindrance of cell membrane alignment during direct cell contact by the blocking Abs has been suggested to possibly confound the results of such in vitro surface molecule blockade (2), and thus the issue remains controversial (7, 10).

The classic pathway for the transmission of signals by soluble TNF-{alpha} involves stimulation of the two receptors TNFR1 and TNFR2. In the signal transmission via mTNF-ligand/TNF-receptor interaction during direct cell contact, a third option is theoretically possible. It can be hypothesized that surface TNFR molecules are able to ligate mTNF on the opposing cell and subsequently trigger signal transduction from the mTNF molecule to the nucleus. Such so-called reverse signaling, the transfer of signals following ligation of membrane-anchored cytokines, has been described for several members of the TNF superfamily including TNF-{alpha} (13, 14, 15, 16, 17, 18). Ligation of mTNF by soluble receptor constructs induces dephosphorylation of the small cytoplasmic part of mTNF, which is crucial for mTNF-mediated calcium signaling (18). In addition, binding of Abs to mTNF can induce apoptosis in several cell types (19, 20, 21). We hypothesized that such reverse signaling transmitted through membrane TNF-{alpha} is involved in the regulation of T cell-dependent monocyte activation in in vitro cocultures. Besides dephosphorylation of the short cytoplasmic tail of mTNF, which is constitutionally phosphorylated by casein kinase, only one other signaling event following ligation mTNF has been described, namely, an increased phosphorylation of the MAPKs ERK1/2, both of which might therefore also be involved in the T cell-dependent monocyte activation by the mTNF-TNFR interaction.

Therefore, the aim of the present study was to analyze the contribution of the membrane TNF/TNF-receptor interaction, including signal transduction through TNFR1/2 and through mTNF, to T cell-induced monocyte activation.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 Disclosures
 References
 
RA patient cohort

Seventeen patients with RA according to the 1987 revised criteria of the American College of Rheumatology were enrolled into the study. The study design was approved by the University of Leipzig’s Ethics Committee, and informed consent was obtained from each patient before study enrollment. After the decision of the rheumatologist taking care of a patient to initiate treatment with the monoclonal anti-TNF-{alpha} Ab Adalimumab (Humira; Abbott Laboratories), pretreatment blood samples were obtained from all patients, and T cell-induced cytokine production by monocytes was determined (see below). After 8 wk of treatment, blood samples were drawn again and the assay was repeated. Clinical documentation of disease activity and response to therapy included the number of swollen and tender joints, duration of morning stiffness, and standard laboratory parameters.

Abs and Materials

Rabbit polyclonal anti-phospho-ERK Ab was purchased from Cell Signaling Technology (Cummings Center, Beverly, MA). Peroxidase-conjugated goat anti-rabbit secondary Ab and anti-GAPDH Ab were purchased from Santa Cruz Biotechnology. Flow cytometry Abs anti-TNF-{alpha} (clone 6402.31), anti-TNFR1 (clone 16803.1), and anti-TNFR2 (clone 22235) and appropriate isotype controls were obtained from R&D Systems. Abs against TNFR2 (clone 22210) and CD45 (clone HI30) for the blocking experiments were purchased from R&D Systems and eBiosciene, respectively. Anti-CD14 Ab was purchased from DakoCytomation. Human IgG (Intraglobin CP) was obtained from Biotest Pharma, TNFR2:Ig Etanercept was obtained from Immunex and anti-TNF-{alpha} Ab infliximab was purchased from Centocor. Fab of infliximab were prepared with an ImmunoPure Fab Preparation Kit from Pierce. Monoclonal anti-CD3{epsilon} Ab for cell culture was purchased from R&D Systems and monoclonal anti-CD28 Ab for cell culture was obtained from BD Biosciences Pharmingen. The ERK inhibitor PD98059 and the casein kinase I inhibitor D4476 were purchased from Calbiochem/Merck Bioscienes).

Cell line

THP-1 cells (human acute monocytic leukemia) were cultured in RPMI 1640 (PAA Laboratories) supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and grown in 5% CO2 at 37°C.

Monocyte isolation

PBMCs were obtained by Ficoll-Paque (Pharmacia Biotech) density gradient centrifugation. After repeated washing in PBS containing EDTA, untouched monocytes were isolated by negative magnetic depletion using hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE Abs (MACS; Miltenyi Biotec) and a magnetic cell separator (MACS) according to the manufacturer’s protocol. The cell preparations were >95% monocytes as determined by morphology and immunofluorescence staining with a mAb against CD14.

Separation and stimulation of human T cells

Human T cells were isolated by counterflow elutriation from PBMCs (Beckman Instruments as described previously (22). CD4+ T cells were isolated by negative magnetic depletion using the MACS system (Miltenyi Biotec) according to the manufacturer’s protocol. The cell preparations were >90% CD4+ T cells. T cells were stimulated with plate-bound anti-CD3{epsilon} Abs and soluble anti-CD28 Abs for 2 days. After stimulation and incubation for 2 days, the cultures contained >90% CD3+ T cells as determined by flow cytometric analysis using a mAb against CD3. Cells were then washed with PBS three times, fixed for 1 min with 0.05% glutaraldehyde, and washed again three times with PBS. This method of cell fixation was shown to inhibit blast transformation and TNF-{alpha} and IL-2 production in response to PMA and ionomycin (data not shown).

Coculture of human monocytes and T cells

Monocytes (1.5 x 106/ml) were cultured in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in 96-well culture plates (Techno Plastic Products) at 37°C and 5% CO2. Fixed T cells were added at a T cell:monocyte ratio of 7:1 and cells were coincubated for 16 h. After incubation, supernatants (200 µl/well, two wells per condition) were harvested and stored at –140°C until cytokine concentration was determined.

Stimulation of monocytes with anti-TNF-{alpha} reagents

Human monocytes (1.5 x 106/ml) were incubated in RPMI 1640 in the presence of 40 µg/ml human IgG or 40 µg/ml soluble TNFR2:IgG construct (Etanercept) for 20 min. Subsequently, cells were rigorously washed with RPMI 1640 to remove these reagents. The cells were placed into RPMI 1640 supplemented with 10% FCS and 2 mM glutamine. After 16 h, supernatant was removed for cytokine measurement. In other experiments, tissue culture plates were coated with human IgG or soluble TNFR2:IgG construct (100 µg/ml) before cell culture. Unbound reagents were removed and monocytes (2 x 106/ml) were incubated in RPMI 1640 supplemented with 10% FCS and 2 mM glutamine in the presence of additional 40 µg/ml soluble human IgG or soluble TNFR2:IgG construct for the indicated times. Subsequently, supernatant was removed for cytokine measurement.

Cytokine secretion assays

Human monocytes (2 x 106/ml) were incubated in RPMI 1640 supplemented with 5% human blood type-matched serum and 2 mM glutamine in the presence of human IgG (100 µg/ml) or soluble TNFR2:IgG construct (100 µg/ml) for 4 h. TNF-{alpha}-producing cells were detected and enriched by commercially available cytokine secretion assays (Miltenyi Biotec) according to the manufacturer’s protocol. Briefly, cells were labeled at 4°C with the catch reagent, a bi-specific Ab-Ab conjugate consisting of an anti-CD45 mAb and an anti-TNF-{alpha} mAb. The catch reagent is anchored to the cell membrane via the binding of CD45 and binds soluble, secreted TNF-{alpha} with its anti-TNF-{alpha} Ab part. After 7 min, cells were transferred to 37°C prewarmed RPMI 1640 at 1 x 106 cells/ml and placed on a rotator at 37°C for 45 min. Secreted TNF-{alpha} binds to the catch reagent. These cells are subsequently labeled with a second anti-TNF-{alpha} Ab conjugated to PE. Magnetic labeling of the cells with anti-PE MicroBeads (Miltenyi Biotec) allows an additional enrichment step. Samples were analyzed on a FACSCalibur by using CellQuest software (BD Biosciences).

Flow cytometry

Cells (2 x 105/50 µl) were incubated with 10% human AB serum to block Fc receptors for 30 min at 4°C. Subsequently, serum was removed and PE-labeled Abs were added. After 30 min at 4°C, cells were washed repeatedly with PBS supplemented with 10% Hemaccel (DeltaSelect) and fixed with 1% formaldehyde. Samples were analyzed on a FACSCalibur by using CellQuest software (BD Biosciences).

Plasmids and small interfering RNA (siRNA)

The cDNA of the entire coding region of wild-type TNF-{alpha} was obtained by PCR from cDNA from LPS-stimulated monocytes with the following primers: 5'-GCTCTAGATGAGCACTGAAAGCATGATCCG-3' and 5'-CGGGATCCTCACAGGGCAATGATCCCAAAG-3'. These primers harbored BamHI and XbaI sites, respectively, to facilitate cloning. The PCR consisted of an initial denaturation at 95°C for 2 min, 35 cycled reactions of 95°C for 60 s, 62°C for 60 s, and 72°C for 90 s, and the final extension at 72°C for 15 min with BioTherm DNA-polymerase (AppliChem). To introduce two mutations (Arg77 and Ser78 to Thr) to minimize cleavage of membrane TNF by TACE, site-directed mutagenesis was used. Both the codon AGA for Arg77 and the codon TCA for Ser78 were substituted to the codon ACA for Thr. Two primers were used to introduce those mutations: forward primer 5'-CAGTCACAACATCTTCTCGAACCC-3' and reverse primer 5'-AAGATGTTGTGACTGCCTGGGCCAGA. In a first step, two PCRs were performed using one mutation-introducing primer and one normal primer. In a second step, the two PCR products were used as templates for a PCR with the two normal primers. The amplified product was cloned into pcDNA3.1 vector at the BamHI/XbaI site (Invitrogen Life Technologies). Plasmids were amplified in Escherichia coli K12DH5{alpha}, identified by complete nucleotide sequencing and purified using an Endofree Plasmid Preparation Kit (Qiagen).

The plasmid containing human full-length TNFR2 was purchased from Origene Technologies. Human TNFR1 and TNFR2 were silenced using a specific siRNA mix containing three different siRNAs provided by Santa Cruz Biotechnology. As a control, siRNA with a nonsense sequence was used.

Transfection of human T cells

Freshly isolated CD4+ T cells were transfected with siRNA or plasmid DNA in a Nucleofector device (Amaxa Biosystems) by using Human T Cell Nucleofector Solution. Briefly, 10 µl of siRNA (10 µM) and/or 2 µg of plasmid DNA was added to 1 x 107 CD4+ T cells, which were resuspended in 100 µl of transfection solution. Cells were subjected to nucleofection using the V-024 program (Amaxa Biosystems). Control cells were transfected with control siRNA and/or control plasmid DNA. To judge transfection efficiency, cells were transfected with 1 of µg pmaxGFP plasmid and GFP fluorescence was determined by FACS. Transfected cells were immediately diluted in 2 ml of 37°C prewarmed RPMI 1640 medium (supplemented with 5% human serum) and seeded into 6-well plates. After 24 or 48 h, cells were harvested and cell viability was determined by the trypan blue exclusion method. Transfected CD4+ T cells were ~50% GFP positive and 90% trypan blue negative. In subsequent experiments, transfected and fixed CD4+ T cells were cocultured with monocytes.

Transfection of THP-1 cells

Monocytic THP-1 cells were transfected with either control nonsense siRNA or specific siRNA targeting TNFR1 or TNFR2 using transfection reagent from Santa Cruz Biotechnology. Briefly, 10 µl of siRNA (10 µM) was added to the transfection reagent and subsequently transferred to 6-well culture plates with 2 x 105/ml THP-1 cells. After 24 h, cells were harvested and cell viability was determined by the trypan blue exclusion method. Transfected THP-1 cells were 95–100% trypan blue negative. In subsequent experiments, transfected cells were cocultured with fixed prestimulated T cells (ratio 7:1) for 4 h, and TNF-{alpha} concentration was determined in the supernatant.

Cell lysis, gel electrophoresis, and Western blotting

Monocytes (3 x 106/2 ml) were stimulated for 15 min with fixed, stimulated T cells or were left untreated. After stimulation, fixed T cells were removed by intensive washing with PBS containing 1 mM sodium orthovanadate. A contamination of monocyte lysates with protein from the T cells is unlikely because most of the T cells were removed by washing, and fixed T cells are not lysed by the lysis buffer used (controlled by protein measurement; data not shown). Cells were lysed in lysis buffer (137 mM NaCl, 50 mM Tris-HCl (pH 7.8) 10% v/v glycerol, 1 mM sodium orthovanadate, 2 mM EDTA (pH 8.0), 1% v/v Nonidet P-40, plus protease inhibitors leupeptin (10 µg/ml) and PMSF (1 mM)) on ice for 15 min. Cell lysates were cleared of debris by centrifugation (10,000 x g, 15 min, 4°C) and protein concentrations of the supernatants were determined with a detergent-compatible protein assay (Bio-Rad). Aliquots of the supernatants were boiled in Laemmli sample buffer for 5 min, and equal amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. Nonspecific binding sites were blocked by incubation in TBS containing 0.5% Tween 20 and 5% w/v dry milk. Subsequently, membranes were incubated with primary Abs at 4°C overnight. Subsequently, membranes were washed three times with TBS and 0.1% Tween 20 at room temperature for 5 min and then incubated with the secondary Ab at room temperature for 1 h. After washing three more times with TBS and 0.1% Tween 20 at room temperature for 5 min, the proteins were visualized by ECL (PerkinElmer) according to the manufacturer’s instructions. Every membrane was used several times to visualize the whole panel of different proteins of interest. Equal protein loading of different lanes was confirmed by visualization with an anti-GAPDH Ab. Between every visualization step, the membranes were stripped with Restore Western Blot Stripping Buffer (Pierce) for 15 min at 37°C, blocked with TBS containing 5% dry milk powder and 0.1% Tween 20 for 1 h, and washed three times with TBS and 0.1% Tween 20 at room temperature for 5 min.

Cytokine measurement

Human TNF-{alpha} (OptEIA Set; BD Biosciences) and IL-8 (Beckman Coulter) was measured by commercially available enzyme immunoassays following the manufacturer’s protocol.

Statistical analysis

For statistical analysis, the software package Sigma Stat (SPSS) was used. Before all comparisons, a normality test was performed. Student’s t test or the Mann-Whitney U rank sum test was used for comparisons where appropriate.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 Disclosures
 References
 
Treatment with anti-TNF-{alpha} Ab reduces T cell-dependent monocyte activation in vitro

Administration of TNF-{alpha}-blocking Ab (anti-TNF-{alpha} Ab) to patients with RA has been shown to reduce disease activity and progression of RA. Because TNF-{alpha} produced by monocytes upon contact with preactivated T cells has been suggested to contribute to the increased TNF-{alpha} levels observed in RA patients, TNF-{alpha} production by monocytes from anti-TNF-{alpha} Ab-treated RA patients coincubated with T cells was measured. Patients received four s.c. injections in biweekly intervals, and the monocyte-T cell coculture assay was conducted 2 wk after the last injection. As shown in Fig. 1, treatment with the anti-TNF-{alpha} Ab leads to a significant decrease in TNF-{alpha} production compared with the pretreatment values. To avoid contamination of the cultures with soluble serum components such as anti-TNF-{alpha} Ab, monocytes were thoroughly washed before they were added to preactivated T cells from healthy donors. Thus, the inhibitory effect of anti-TNF-{alpha} treatment is likely due to a direct interference of the Ab with the monocyte-T cell interaction.


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 1. Treatment with an anti-TNF-{alpha} mAb inhibits T cell-induced TNF-{alpha} production by monocytes from RA patients. Peripheral blood T cells from healthy individuals (1 x 106/ml) were cultured for 48 h in the presence (sTc) or absence (rTc) of immobilized anti-CD3 (3.3 µg/ml) and soluble anti-CD28 (0.8 µg/ml) Abs. T cells were fixed and incubated with freshly isolated monocytes from RA patients (1.5 x 106/ml) at a ratio of 7:1 for 16 h. Values are means ± SEM of TNF-{alpha} production by monocytes isolated from 17 RA patients before (gray bars) and during treatment (dark gray bars).

 
Potential interaction partners that could be affected by the anti-TNF-{alpha} Abs are transmembrane TNF-{alpha} (mTNF) and the TNFRs 1 and 2 (TNFR1/R2).

Transmembrane TNF-{alpha} on activated T cells induces monocyte activation during direct cell contact

To test whether mTNF and TNFR1 and 2 are expressed on T cells and monocytes, FACS analysis was conducted. In healthy controls, mTNF was expressed in detectable amounts on the cell surface of freshly isolated monocytes and prestimulated T cells (Fig. 2a), whereas TNFR2 was expressed on both monocytes and activated T cells. TNFR1 expression was only found on monocytes, but not on activated T cells (Fig. 2a). The expression of TNFR1 and TNFR2 did not differ between RA patients treated with conventional DMARDs (mean fluorescence, TNFR1 54.9 ± 20.3, TNFR2 77.7 ± 6.3, n = 8) compared with anti-TNF-{alpha}-treated patients (mean fluorescence TNFR1 50.3 ± 16.5, TNFR2 84.9 ± 15.4, n = 8; data not shown). In contrast to prestimulated T cells, resting T cells did not express mTNF (Fig. 2b).


Figure 2
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 2. Expression of transmembrane TNF-{alpha}, TNFR1, and TNFR2 on activated T cells and monocytes. a, Cell surface expression of mTNF{alpha}, TNFR1, and TNFR2 was determined by flow cytometry on freshly isolated monocytes and on T cells stimulated with anti-CD3 Ab (see Patients and Methods). Shown are representative histograms from one healthy donor of eight. b, mTNF{alpha} is not expressed on resting T cells, but is up-regulated upon activation. Peripheral blood CD4+ T cells (1 x 106/ml) were cultured for 48 h in the presence (sTc) or absence (rTc) of immobilized anti-CD3 (3.3 µg/ml) and soluble anti-CD28 (0.8 µg/ml) Abs. Bar charts depict means ± SEM of experiments with eight different donors. Histogram shows mTNF{alpha} expression on resting and stimulated T cells from one representative healthy donor of eight.

 
To examine the role of transmembrane TNF-{alpha} on the surface of preactivated T cells in the activation of monocytes leading to TNF-{alpha} production, stimulated T cells were preincubated with anti-TNF-{alpha} Ab and extensively washed and fixed before monocytes were added. Pretreatment of T cells with the mAb led to a significant reduction in TNF-{alpha} production (968 ± 243 pg/ml vs 152 ± 109 pg/ml, p = 0.037, n = 3; data not shown). Subsequently, Fab of the anti-TNF-{alpha} Ab were used instead of the complete Ab to avoid in vitro stimulation of monocytes by the Fc fragments. The results obtained with the Fab did not differ from those obtained with the complete Ab and showed an abrogation of the monocyte TNF-{alpha} response (Fig. 3a).


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 3. Blockade of transmembrane TNF-{alpha} on stimulated T cells inhibits TNF-{alpha} production by monocytes. Peripheral blood T cells (1 x 106/ml) were cultured for 48 h in the presence of immobilized anti-CD3 (3.3 µg/ml) and soluble anti-CD28 (0.8 µg/ml) Abs. T cells were washed and incubated for 30 min at 4°C with the Fab of anti-TNF-{alpha} Ab infliximab (1 µg/ml) to block transmembrane TNF-{alpha}. Subsequently, T cells were washed with PBS to remove unbound Abs, fixed, and incubated with freshly isolated monocytes (1.5 x 106/ml) at a ratio of 7:1. Concentrations of TNF-{alpha} (a) or IL-8 (b) were measured after 16 h in the supernatant. Bar charts depict means ± SEM of results from four independent experiments. c and d, T cells stimulated as described above were added to monocytes at a ratio of 7:1. Freshly isolated monocytes were incubated for 15 min in culture medium only (control), in the presence of stimulated T cells (sTc) or in the presence of stimulated T cells with blocked transmembrane TNF (sTc [anti-TNF-{alpha}]). Phosphorylation of ERK MAPK in monocytes was determined by Western blot analysis as described in Patients and Methods. Shown is one representative blot of three (c) and intensity read outs (d).

 
Although the anti-TNF-{alpha} Ab binds mTNF with high affinity, which is further enhanced by fixation with glutaraldehyde, dissociation of the bound Ab can still occur and may lead to the release of soluble anti-TNF-{alpha} Ab into the cocultures. The soluble Ab could interfere with the detection of TNF-{alpha} by ELISA, so that the measured concentrations are falsely low. To verify the inhibitory effect of the anti-TNF-{alpha} Ab treatment, another cytokine, namely, IL-8, which is also produced by monocytes in the coculture system, was measured. IL-8 was chosen as a read-out system because the neutralizing anti-TNF-{alpha} Ab did not block LPS-induced IL-8 production, indicating that the secretion of this cytokine was independent of the autocrine TNF-{alpha} production (data not shown). Concentrations of IL-8 were profoundly reduced when mTNF was blocked on the stimulated T cells (Fig. 3b).

The MAPK pathways, and in particular ERK 1/2, have been shown to participate in the signal transduction in monocytes during contact with activated T cells (5, 23) and after ligation of TNFRs (24). Accordingly, addition of the ERK inhibitor PD98059 to cocultures of monocytes and activated T cells led to a decreased T cell-dependent TNF-{alpha} production by monocytes (data not shown). When phosphorylation of ERK 1/2 was analyzed in monocytes cocultured with preactivated T cells and compared with controls, a strong phosphorylation signal was detected (Fig. 3c). Blockade of mTNF on preactivated T cells by preincubation with anti-TNF-{alpha} Ab led to a reduction of this phosphorylation (100% vs 71.7% ± 7.2; p = 0.017; Fig. 3, c and d), confirming the stimulatory interaction between mTNF on T cells and TNFRs on monocytes.

To confirm that monocytes are stimulated via mTNF, freshly isolated CD4+ T cells were transfected with an expression plasmid containing an uncleavable form of TNF-{alpha}. The expressed TNF-{alpha} was membrane anchored and could be detected by flow cytometry. Surface mTNF was present on ~30% of the transfected CD4+ T cells, but not on mock-transfected cells (Fig. 4a).


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 4. Transfected mTNF expressed on the surface of resting T cells induces TNF-{alpha} production in monocytes. Resting CD4+ T cells (rTc) were transfected with pcDNA3.1 mTNF (mTNF plasmid) or transfected with the empty pcDNA3.1 vector (control plasmid) and subsequently cultured for 1 day. a, Dot blot depicts the percentage of mTNF-positive CD4+ T cells in the cultures following the transfection. The quadrant was set according to the isotype control. Shown is one representative experiment of three. b, Transfected resting CD4+ T cells were fixed and incubated with freshly isolated monocytes (1.5 x 106/ml) at a ratio of 7:1. After 16 h, the concentration of TNF-{alpha} was measured in the supernatant. Data are means ± SEM of values from three independent experiments.

 
The transfected cells were fixed and subsequently used in the coculture system with monocytes. mTNF-transfected CD4+ T cells induced a strong TNF-{alpha} production in monocytes, while mock-transfected CD4+ T cells did not (Fig. 4b).

Down-regulation of TNFRs on monocytes reduces TNF-{alpha} response in the coculture assay

To test whether mTNF on T cells reacts with TNFR1 and 2 on monocytes and to distinguish between the individual contributions of the two TNFRs to cell contact-dependent activation of monocytes, selective blockade of the receptors was necessary. The used coculture system requires coincubation of monocytes with T cells over 16 h. The high turnover and the rapid internalization of the two TNFRs upon ligand binding (25, 26) makes the use of receptor-blocking Abs unreliable. Newly expressed receptor molecules that emerge on the cell surface during the prolonged incubation period may not be blocked sufficiently. Thus, instead of blocking Abs, specific siRNAs were used to modulate the surface expression of TNFR1 and TNFR2 on the monocytic cell line THP-1. THP-1 cells were chosen because they express both TNFRs and produce TNF-{alpha} after cell contact with stimulated T cells (data not shown).

Transfection of THP-1 cells with TNFR1 siRNA significantly inhibited TNFR1 expression and resulted in a decrease of TNFR1-positive THP1 cells compared with THP-1 cells transfected with nonsense siRNA (35.7 ± 1.3% vs 26.7 ± 1.2%; p = 0.007; Fig. 5a). Similarly, transfection with TNFR2 inhibited surface expression of TNFR2 and decreased the percentage of TNFR2-positive THP-1 cells after transfection (34.9 ± 2.0% vs 23.5 ± 3.3%; p = 0.043; Fig. 5a). In contrast, transfection with TNFR1 siRNA had no influence on TNFR2 expression and vice versa (data not shown).


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 5. Inhibition of TNFR1 and 2 expression on THP1 cells results in decreased TNF-{alpha} production by monocytes in response to direct cell contact. a, Transfection of THP-1 cells with TNFR1 or TNFR2 siRNA significantly decreased the percentage of TNFR1-positive or TNFR2-positive THP-1 cells. As controls, THP-1 cells were electroporated with nonsense siRNA. Data are means of TNFR-positive cells ± SEM of values from three independent experiments. b, Peripheral blood T cells (1 x 106/ml) were activated with immobilized anti-CD3 and soluble anti-CD28 Abs, fixed, and cocultured with THP-1 cells (1.5 x 106/ml) for 4 h at a ratio of 7:1. Bar charts depict TNF-{alpha} concentrations in the supernatant as percentage of the TNF-{alpha} concentration produced in the control wells. Values are means ± SEM of three independent experiments.

 
Following transfection, the THP-1 cells were used in the coculture assay, and TNF-{alpha} production elicited by direct cell contact with activated T cells was determined. Down-regulation of surface expression of both TNFR1 and TNFR2 was found to be associated with a significant decrease in the TNF-{alpha} production of THP-1 cells in coculture with activated T cells (Fig. 5b) compared with cocultures of T cells with THP-1 cells transfected with nonsense siRNA.

Reverse signaling of mTNF on monocyte surfaces contributes to the T cell-dependent TNF-{alpha} response in the coculture assay

So far we have shown that mTNF molecules on activated T cells are interacting with TNFR1 and TNFR2 molecules on peripheral blood monocytes, thereby triggering an activation signal. It cannot be excluded, however, that mTNF on monocytes participates in signal transduction by a mechanism called reverse signaling (Fig. 9a). For several membrane-anchored cytokines, such signal transduction mechanisms have been described previously (13, 14, 15, 16, 17, 18). To explore the contribution of reverse mTNF signaling in monocytes to T cell-dependent TNF-{alpha} production, the potential counterpart of mTNF, namely, TNFR2, on the T cell surface was blocked. When stimulated T cells were incubated with anti-TNFR2 Ab at 4°C, fixed, and subsequently used in the coculture assay, monocyte TNF-{alpha} production was significantly inhibited in contrast to the control Ab against CD45 (Fig. 6a).


Figure 9
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 9. Schematic model of mTNF-TNFR interactions during cell contact between monocytes and T cells. a, Through cellular contact, TNFR1 and TNFR2 on the monocyte surface are stimulated by binding mTNF on fixed T cells, which results in the triggering of an activating signal to the monocytes. Simultaneously, TNFR2 molecules on the surface of the activated T cells ligate mTNF on monocytes, thereby triggering reverse signaling into the monocytes, which contributes to their activation during cell contact with activated T cells. b, Hypothetical model of the in vivo situation in RA patients treated with a monoclonal anti-TNF-{alpha} Ab. In this scenario, monocytic TNF-{alpha} production in RA is elicited by direct cell contact with pathologically activated T cells. After administration of the therapeutic Ab, mTNF is blocked on T cells and monocytes, resulting in simultaneous inhibition of signaling through TNF receptors and reverse signaling via mTNF.

 

Figure 6
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 6. Blockade of TNFR2 on activated T cells reduces the T cell-induced monocyte activation. a, Peripheral blood T cells (1 x 106/ml) were activated with immobilized anti-CD3 and soluble anti-CD28 Abs and subsequently incubated for 30 min at 4°C with anti-TNFR2 Ab (10 µg/ml) (sTc [a-TNFR2]) or anti-CD45 Ab (10 µg/ml) (sTc [a-CD45]) as isotype control. After rigorous washing to remove unbound Ab, T cells were fixed and incubated with freshly isolated monocytes (1.5 x 106/ml) at a ratio of 7:1. After 16 h, the concentration of TNF-{alpha} was measured in the supernatant. Data are means ± SEM of values from three independent experiments. b and c, Freshly isolated monocytes (3 x 106/2 ml) at a ratio of 7:1 were incubated without T cells (control), with stimulated T cells (sTc), or with stimulated T cells preincubated with anti-TNFR2 Ab (sTc [a-TNFR2]) or stimulated T cells preincubated with anti-CD45 Ab (sTc [a-CD45]) as isotype control. After 15 min, cells were lysed, and phosphorylation of ERK MAPK was determined by Western blotting. GAPDH protein levels were used as loading controls. Shown is one representative experiment of three (b) and intensity read-outs (c).

 
Western blot analysis of ERK 1/2 phosphorylation revealed that blockade of TNFR2 on the stimulated T cells leads to a decrease of phosphorylation (100% vs 44.3% ± 7.2%; p = 0.016, Fig. 6, b and c), which confirms the stimulatory effect of mTNF ligation by TNFR2.

To selectively analyze the effect of reverse signaling of mTNF on monocyte surfaces, which is elicited by binding to the TNFR, TNFR2 was overexpressed on freshly isolated CD4+ T cells. Circulating, unstimulated T cells express only low amounts of TNFR2 and induce only minimal TNF-{alpha} production in monocytes (99 ± 33 pg/ml vs medium control 13 ± 6 pg/ml, p = 0.026). Transfection of freshly isolated CD4+ T cells with a plasmid containing TNFR2 cDNA led to high expression of TNFR2 (Fig. 7a), comparable to that seen on activated T cells (Fig. 2a). In contrast to activated T cells, however, no expression of mTNF was detectable on transfected T cells (data not shown). Transfected CD4+ T cells were fixed and cocultured with monocytes. In accordance with the mTNF reverse signaling hypothesis, monocytes were found to produce high levels of TNF-{alpha} in coculture with TNFR2-overexpressing CD4+ T cells, but not with CD4+ T cells transfected with the control plasmid (Fig. 7b).


Figure 7
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 7. T cells transfected with TNFR2 induce a strong response in monocytes during direct cell contact, while this response can be suppressed by down-regulation of TNFR2. a and b, Resting CD4+ T cells (rTc) were transfected with pcDNA3.1 TNFR2 (TNFR2 plasmid) or with the empty pcDNA3.1 vector (control plasmid) and subsequently cultured for 2 days. a, Dot blot depicts expression of TNFR2 on CD4+ T cells in the cultures following transfection. The quadrant was set according to the isotype control, and percentages of TNFR2-positive cells are given. Shown is one representative dot blot of three. b, Transfected, unstimulated CD4+ T cells were fixed and incubated with freshly isolated monocytes (1.5 x 106/ml) at a ratio of 7:1. After 16 h, the concentration of TNF-{alpha} was measured in the supernatant. Data are means ± SEM of values from three independent experiments. c and d, Resting CD4+ T cells (rTc) were transfected with pcDNA3.1 mTNF (mTNF plasmid) and simultaneously with either TNFR2 siRNA (mTNF(+)TNFR2(–)) or nonsense siRNA (mTNF(+)TNFR2(+)) as a control, followed by culture for 1 day. c, Dot blot depicts the TNFR2 expression on T cells following transfection, whereas the quadrant is adjusted to the isotype control. Shown is one representative dot blot of three. d, Transfected CD4+ T cells were fixed and incubated with freshly isolated monocytes (1.5 x 106/ml) at a ratio of 7:1 for 16 h. Bar charts depict TNF-{alpha} concentrations in the supernatant as percentage of the TNF-{alpha} concentration produced in the cocultures of monocytes with transfected T cells. Data are means ± SEM of the values from three independent experiments.

 
To evaluate the respective contribution of membrane TNF and TNFR2 on T cells in the interaction with monocytes, freshly isolated CD4+ T cells were transfected with a plasmid containing an uncleavable form of TNF-{alpha}. Experiments with simultaneous transfection of mTNF and TNFR2 were not possible due to the different kinetics of membrane expression of the two molecules and a severely reduced viability of the cotransfected cells. Instead, we took advantage of the low, but reproducibly detectable expression of TNFR2 on freshly separated CD4+ T cells by selectively down-regulating it. After transfection with mTNF, the CD4+ T cells were simultaneously cotransfected with TNFR2-targeting siRNA, which led to the abrogation of the spontaneous expression of this protein (Fig. 7c). The double-transfected CD4+ T cells were subsequently fixed and cocultured with monocytes. In the presence of constitutively expressed TNFR2 and transfected, uncleavable surface mTNF on T cells, high levels of TNF were induced in the monocytes (Fig. 7d). Inhibition of surface TNFR2 expression on the T cells transfected with mTNF resulted in a reduction of the induced TNF levels by 50% (Fig. 7d).

In an additional experiment investigating the effect of reverse signaling through mTNF on monocytes, purified cells were incubated with a soluble TNFR2:IgG construct in the absence of T cells. Addition of TNFR2:Ig to freshly isolated peripheral blood monocytes from healthy donors resulted in a low but detectable TNF-{alpha} production of the monocytes after 16 h of culture (Fig. 8a). This low TNF-{alpha} production was enhanced when monocytes were incubated in tissue culture plates coated with TNFR2:Ig. The resulting cross-linking of mTNF on monocytes by plate-bound TNFR2 led to the production of high levels of TNF-{alpha} (Fig. 8b). This TNFR2:Ig-induced TNF-{alpha} production was found to be also dependent on MAPK pathways, because it was reduced when ERK activity was inhibited by PD98059 (Fig. 8c). When casein kinase I, the enzyme responsible for mTNF phosphorylation, was inhibited, the TNF-{alpha} production was also found to be decreased (Fig. 8c). Furthermore, using a highly sensitive commercial cytokine secretion assay, production of TNF-{alpha} following incubation of monocytes with TNFR2:Ig was also clearly detectable (Fig. 8d).


Figure 8
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 8. Ligation of mTNF{alpha} by TNFR2:Ig induces TNF-{alpha} production in monocytes. a, Monocytes (1.5 x 106/ml) were incubated in the presence of 40 µg/ml Etanercept (TNFR2:Ig) or human IgG for 20 min. After rigorous washing to remove the reagents, monocytes were incubated for 16 h and concentrations of TNF-{alpha} were measured in the supernatant. Data are means ± SEM of values from 11 independent experiments. b, Monocytes (2 x 106/ml) were incubated on tissue culture plates coated either with TNFR2:Ig (Etanercept) or with human IgG as a control. Bar chart depicts the concentration of TNF-{alpha} in the supernatant at the time points indicated. Data are means ± SEM of values from three (4 h, 16 h) or four (24 h) independent experiments. c, Monocytes (2 x 106/ml) were incubated on tissue culture plates coated with soluble TNFR2:Ig construct (TNFR2:Ig) for 16 h in the absence (control) or presence of 10 µM of the ERK inhibitor PD98059 (PD) or 150 µM of the casein kinase I inhibitor D4476. Bar chart depicts TNF-{alpha} concentrations in the supernatant as percentage of the TNF-{alpha} concentration produced in cultures without the inhibitor. Data are means ± SEM of values from three independent experiments. d, Detection of TNF-{alpha}-producing monocytes after reverse signaling through mTNF{alpha}. Monocytes (2 x 106/ml) were incubated in the presence of 100 µg/ml Etanercept (TNFR2:Ig) or human IgG for 4 h. After rigorous washing, TNF-{alpha}-producing cells were detected using a commercial cytokine secretion assay (see Patients and Methods). Histograms depict the fluorescence intensity of the anti-TNF-{alpha} detection Ab on monocytes after the immunomagnetic enrichment step. The marker indicates the percentage of TNF-{alpha}-producing cells. Shown is one representative experiment of five.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
Disclosures
 References
 
T cell contact-induced cytokine production by monocytes has previously been reported to involve ligand receptor pairs anchored in the opposing membranes of the two cell types (4, 5, 7, 8). In this study, we present evidence that the interaction between mTNF and its two receptors TNFR1 and TNFR2 plays a predominant role in this cell contact-dependent stimulation. We show that blocking mTNF on the surface of activated T cells almost completely abrogates monocyte activation and that in contrast mTNF-transfected T cells induce activation of monocytes.

mTNF on T cells triggers stimulatory signals in the monocytes via both receptors, TNFR1 and TNFR2, as demonstrated by the down-regulation of the two receptors in monocytic cells by the use of siRNAs. Both siRNAs were equally effective in reducing TNF-{alpha} production, indicating that both receptors are involved in this stimulation and that a certain degree of redundancy in the function of the two receptors is likely.

After binding of TNF-{alpha} to the two receptors, initiation of several different cellular signal transduction pathways has been described.

TNFR1 appears to be the key mediator of signaling of soluble trimerized TNF-{alpha}. Binding of TNF-{alpha} to TNFR1 initiates the apoptotic signaling cascade which leads to the formation of the death-inducing signaling complex (DISC) and the induction of cellular apoptosis via recruitment of caspase 8 to the DISC. For this pathway to operate, internalization of the complex consisting of soluble TNF-{alpha} and TNFR1 and formation of TNF receptosomes as death-signaling vesicles are required (27).

However, because internalization of a complex consisting of mTNF (on T cells) and TNFR1 into the monocytes can be excluded, the alternative signal transduction pathway described for TNFR1 is likely to be induced. This pathway, which involves recruitment of TNFR1 molecules to lipid rafts and initiates the formation of a signaling complex containing receptor-interacting protein, TNFR-associated death domain protein and TNFR-associated factor 2 (28), results in cellular activation and is most likely to be initiated in the here described way of monocyte activation.

Signaling of TNFR2 upon binding of TNF-{alpha} is also associated with TNFR-associated factor 2 recruitment (29) and seems to use overlapping signaling pathways resulting in cellular activation.

Downstream signaling of both TNFRs includes activation of the three major MAPK families, ERK, c-Jun amino-terminal kinases (JNK), and p38 MAPK{alpha}. The observed ERK phosphorylation in monocytes cocultured with activated T cells is therefore consistent with the hypothesis of a major role of the mTNF-TNFR interaction.

In the clinical study presented in the first section of Results, we have shown that monocytes from RA patients treated with anti-TNF-{alpha} Ab produced less TNF-{alpha} in the coculture system compared with the pretreatment values. Several explanations can be excluded due to the experimental conditions. 1) Transfer of soluble TNF-{alpha} or anti-TNF-{alpha} Ab to the cultures does not occur, because no soluble serum components were added to the coculture system. 2) Blockade of mTNF on T cells can be excluded because activated T cells from healthy donors were used. 3) Down-regulation of TNFR expression on monocytes during therapy is not likely, because addition of TNF-{alpha} rather than blockade of the cytokine has been shown to result in the down-regulation of both TNFRs at least in vitro (30). In addition, expression levels of TNF receptors on monocytes of RA patients receiving TNF inhibitor therapy did not differ significantly from methotrexate-treated patients in a retrospective, cross-sectional study. As an alternative mechanism responsible for the observed changes, we investigated reverse signaling through mTNF on monocytes.

We could show that signaling through mTNF indeed results in cytokine production by monocytes. Blocking TNFR2, the potential interaction partner on T cells, resulted in a decrease of TNF-{alpha} production by monocytes. Interaction of TNFR2 molecules on transfected and fixed T cells with monocytes induces cytokine production. This interaction also partially contributes to the response elicited in monocytes during cell contact with activated T cells, since suppression of TNFR2 expression on these T cells decreases this response. Furthermore, ligation of mTNF by the soluble TNFR2 construct induced TNF-{alpha} production by monocytes, which was detectable in the supernatant by ELISA and in a highly sensitive flow cytometric secretion assay. Cross-linking of mTNF molecules on monocytes using plate-bound TNFR2:Ig construct, which is likely to mimic the effect of direct cell contact more closely, further enhanced the TNF-{alpha} production elicited by TNF reverse signaling.

Following reverse signaling through mTNF, the induction of two different pathways leading to different cellular responses has been described. An increase in intracellular calcium concentrations following mTNF ligation has been related to cellular apoptosis due to autocrine TGF-beta production or direct cellular signaling (18, 31). However, according to a more recent study, ligation of mTNF by anti-TNF-{alpha} Ab not only induces apoptosis but also the production of IL-10 (32). Furthermore, Waetzig et al. (33) described an up to a 6-fold increase in TNF-{alpha} mRNA expression and activation of p38 in monocytes following ligation of mTNF. These latter data are in line with the results presented here showing that signaling via mTNF results in TNF-{alpha} production. In the T cell:monocyte coculture system used in our experiments, ERK1/2 rather than p38 appears to be the dominant signal transduction pathway, however, since no significant increase in p38 phosphorylation following direct T cell contact or mTNF ligation by plate-bound TNFR2 occurs (data not shown). Interestingly, a strong increase in circulating TNF-{alpha} concentration was observed shortly after administration of a single dose of infliximab in RA (34), and reverse signaling of mTNF might contribute to this increase.

In conclusion, we have shown that as a result of mTNF-TNFR interaction, monocytes receive two signals during contact with activated T cells (Fig. 9a). The first signal is triggered by mTNF on T cell membranes and transduced to the monocytes through both TNFRs. The second signal is elicited when TNFR2 molecules on T cells ligate mTNF on monocytes during cell contact. The signal is transmitted into the monocytes through reverse signaling of mTNF and can be blocked in vitro by anti-TNF-{alpha} Ab. Administration of anti-TNF-{alpha} Ab to RA patients is likely to block both pathways in vivo, which in turn may contribute to the clinical effects seen in those patients (Fig. 9b).


   Disclosures
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The work presented in this study was supported by grants from the German Ministry for Education and Science (Interdisziplinäres Zentrum für Klinische Forschung Leipzig, Teilprojekt A 21 and Kompetenznetzwerk Rheuma, Entzündlich-rheumatische Systemerkrankungen, Teilprojekt C2.7). Back

2 Address correspondence and reprint requests to Dr. Ulf Wagner, Department of Medicine IV, University of Leipzig, Liebigstrasse 22, Leipzig, Germany. E-mail address: wagu{at}medizin.uni-leipzig.de Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; mTNF, transmembrane TNF; siRNA, small interfering RNA. Back

Received for publication July 5, 2006. Accepted for publication July 2, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Burger, D., J. M. Dayer. 2002. The role of human T-lymphocyte-monocyte contact in inflammation and tissue destruction. Arthritis Res. 4: (Suppl. 3):S169-S176. [Medline]
  2. McInnes, I. B., B. P. Leung, F. Y. Liew. 2000. Cell-cell interactions in synovitis: interactions between T lymphocytes and synovial cells. Arthritis Res. 2: 374-378. [Medline]
  3. Brennan, F. M., A. L. Hayes, C. J. Ciesielski, P. Green, B. M. Foxwell, M. Feldmann. 2002. Evidence that rheumatoid arthritis synovial T cells are similar to cytokine-activated T cells: involvement of phosphatidylinositol 3-kinase and nuclear factor {kappa}B pathways in tumor necrosis factor {alpha} production in rheumatoid arthritis. Arthritis Rheum. 46: 31-41. [Medline]
  4. McInnes, I. B., B. P. Leung, R. D. Sturrock, M. Field, F. Y. Liew. 1997. Interleukin-15 mediates T cell-dependent regulation of tumor necrosis factor-{alpha} production in rheumatoid arthritis. Nat. Med. 3: 189-195. [Medline]
  5. Foey, A. D., M. Feldmann, F. M. Brennan. 2001. CD40 ligation induces macrophage IL-10 and TNF-{alpha} production: differential use of the PI3K and p42/44 MAPK-pathways. Cytokine 16: 131-142. [Medline]
  6. Burger, D., N. Molnarfi, L. Gruaz, J. M. Dayer. 2004. Differential induction of IL-1beta and TNF by CD40 ligand or cellular contact with stimulated T cells depends on the maturation stage of human monocytes. J. Immunol. 173: 1292-1297. [Abstract/Free Full Text]
  7. Isler, P., E. Vey, J. H. Zhang, J. M. Dayer. 1993. Cell surface glycoproteins expressed on activated human T cells induce production of interleukin-1beta by monocytic cells: a possible role of CD69. Eur. Cytokine Network 4: 15-23. [Medline]
  8. Manie, S., J. Kubar, M. Limouse, B. Ferrua, M. Ticchioni, J. P. Breittmayer, J. F. Peyron, L. Schaffar, B. Rossi. 1993. CD3-stimulated Jurkat T cells mediate IL-1beta production in monocytic THP-1 cells: role of LFA-1 molecule and participation of CD69 T cell antigen. Eur. Cytokine Network 4: 7-13. [Medline]
  9. Chizzolini, C., R. Chicheportiche, D. Burger, J. M. Dayer. 1997. Human Th1 cells preferentially induce interleukin (IL)-1beta while Th2 cells induce IL-1 receptor antagonist production upon cell/cell contact with monocytes. Eur. J. Immunol. 27: 171-177. [Medline]
  10. Lacraz, S., P. Isler, E. Vey, H. G. Welgus, J. M. Dayer. 1994. Direct contact between T lymphocytes and monocytes is a major pathway for induction of metalloproteinase expression. J. Biol. Chem. 269: 22027-22033. [Abstract/Free Full Text]
  11. Parry, S. L., M. Sebbag, M. Feldmann, F. M. Brennan. 1997. Contact with T cells modulates monocyte IL-10 production: role of T cell membrane TNF-{alpha}. J. Immunol. 158: 3673-3681. [Abstract]
  12. Dai, S. M., H. Matsuno, H. Nakamura, K. Nishioka, K. Yudoh. 2004. Interleukin-18 enhances monocyte tumor necrosis factor {alpha} and interleukin-1beta production induced by direct contact with T lymphocytes: implications in rheumatoid arthritis. Arthritis Rheum. 50: 432-443. [Medline]
  13. Tanabe, Y., C. Kohchi, N. Kitahara-Tanabe, D. Mizuno, G. Soma. 1998. Involvement of 26-kDa membrane-bound tumour necrosis factor precursor in bidirectional feedback regulation on 17-kDa tumour necrosis factor production after stimulation by lipopolysaccharide. Cytokine 10: 82-92. [Medline]
  14. Pocsik, E., E. Duda, D. Wallach. 1995. Phosphorylation of the 26 kDa TNF precursor in monocytic cells and in transfected HeLa cells. J. Inflamm. 45: 152-160. [Medline]
  15. Domonkos, A., A. Udvardy, L. Laszlo, T. Nagy, E. Duda. 2001. Receptor-like properties of the 26 kDa transmembrane form of TNF. Eur. Cytokine Network 12: 411-419. [Medline]
  16. Harashima, S., T. Horiuchi, N. Hatta, C. Morita, M. Higuchi, T. Sawabe, H. Tsukamoto, T. Tahira, K. Hayashi, S. Fujita, Y. Niho. 2001. Outside-to-inside signal through the membrane TNF-{alpha} induces E-selectin (CD62E) expression on activated human CD4+ T cells. J. Immunol. 166: 130-136. [Abstract/Free Full Text]
  17. Eissner, G., S. Kirchner, H. Lindner, W. Kolch, P. Janosch, M. Grell, P. Scheurich, R. Andreesen, E. Holler. 2000. Reverse signaling through transmembrane TNF confers resistance to lipopolysaccharide in human monocytes and macrophages. J. Immunol. 164: 6193-6198. [Abstract/Free Full Text]
  18. Watts, A. D., N. H. Hunt, Y. Wanigasekara, G. Bloomfield, D. Wallach, B. D. Roufogalis, G. Chaudhri. 1999. A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in "reverse signalling.". EMBO J. 18: 2119-2126. [Medline]
  19. Lugering, A., M. Schmidt, N. Lugering, H. G. Pauels, W. Domschke, T. Kucharzik. 2001. Infliximab induces apoptosis in monocytes from patients with chronic active Crohn’s disease by using a caspase-dependent pathway. Gastroenterology 121: 1145-1157. [Medline]
  20. ten Hove, T., C. van Montfrans, M. P. Peppelenbosch, S. J. van Deventer. 2002. Infliximab treatment induces apoptosis of lamina propria T lymphocytes in Crohn’s disease. Gut 50: 206-211. [Abstract/Free Full Text]
  21. Waetzig, G. H., P. Rosenstiel, A. Arlt, A. Till, K. Brautigam, H. Schafer, S. Rose-John, D. Seegert, S. Schreiber. 2005. Soluble tumor necrosis factor (TNF) receptor-1 induces apoptosis via reverse TNF signaling and autocrine transforming growth factor-beta1. FASEB J. 19: 91-93. [Abstract/Free Full Text]
  22. Rossol, M., S. Kaltenhauser, R. Scholz, H. Hantzschel, S. Hauschildt, U. Wagner. 2005. The contact-mediated response of peripheral-blood monocytes to preactivated T cells is suppressed by serum factors in rheumatoid arthritis. Arthritis Res. Ther. 7: R1189-R1199. [Medline]
  23. Harigai, M., M. Hara, M. Kawamoto, Y. Kawaguchi, T. Sugiura, M. Tanaka, M. Nakagawa, H. Ichida, K. Takagi, S. Higami-Ohsako, et al 2004. Amplification of the synovial inflammatory response through activation of mitogen-activated protein kinases and nuclear factor {kappa}B using ligation of CD40 on CD14+ synovial cells from patients with rheumatoid arthritis. Arthritis Rheum. 50: 2167-2177. [Medline]
  24. MacEwan, D. J.. 2002. TNF receptor subtype signalling: differences and cellular consequences. Cell. Signal. 14: 477-492. [Medline]
  25. Yoshie, O., K. Tada, N. Ishida. 1986. Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors. J. Biochem. 100: 531-541. [Abstract/Free Full Text]
  26. Watanabe, N., H. Kuriyama, H. Sone, H. Neda, N. Yamauchi, M. Maeda, Y. Niitsu. 1988. Continuous internalization of tumor necrosis factor receptors in a human myosarcoma cell line. J. Biol. Chem. 263: 10262-10266. [Abstract/Free Full Text]
  27. Schneider-Brachert, W., V. Tchikov, J. Neumeyer, M. Jakob, S. Winoto-Morbach, J. Held-Feindt, M. Heinrich, O. Merkel, M. Ehrenschwender, D. Adam, et al 2004. Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity 21: 415-428. [Medline]
  28. Legler, D. F., O. Micheau, M. A. Doucey, J. Tschopp, C. Bron. 2003. Recruitment of TNF receptor 1 to lipid rafts is essential for TNF{alpha}-mediated NF-{kappa}B activation. Immunity 18: 655-664. [Medline]
  29. Reinhard, C., B. Shamoon, V. Shyamala, L. T. Williams. 1997. Tumor necrosis factor {alpha}-induced activation of c-Jun N-terminal kinase is mediated by TRAF2. EMBO J. 16: 1080-1092. [Medline]
  30. Higuchi, M., B. B. Aggarwal. 1994. TNF induces internalization of the p60 receptor and shedding of the p80 receptor. J. Immunol. 152: 3550-3558. [Abstract]
  31. Higuchi, M., K. Nagasawa, T. Horiuchi, M. Oike, Y. Ito, M. Yasukawa, Y. Niho. 1997. Membrane tumor necrosis factor-{alpha} (TNF-{alpha}) expressed on HTLV-I-infected T cells mediates a costimulatory signal for B cell activation: characterization of membrane TNF-{alpha}. Clin. Immunol. Immunopathol. 82: 133-140. [Medline]
  32. Mitoma, H., T. Horiuchi, N. Hatta, H. Tsukamoto, S. Harashima, Y. Kikuchi, J. Otsuka, S. Okamura, S. Fujita, M. Harada. 2005. Infliximab induces potent anti-inflammatory responses by outside-to-inside signals through transmembrane TNF-{alpha}. Gastroenterology 128: 376-392. [Medline]
  33. Waetzig, G. H., D. Seegert, P. Rosenstiel, S. Nikolaus, S. Schreiber. 2002. p38 mitogen-activated protein kinase is activated and linked to TNF-{alpha} signaling in inflammatory bowel disease. J. Immunol. 168: 5342-5351. [Abstract/Free Full Text]
  34. Charles, P., M. J. Elliott, D. Davis, A. Potter, J. R. Kalden, C. Antoni, F. C. Breedveld, J. S. Smolen, G. Eberl, K. deWoody, et al 1999. Regulation of cytokines, cytokine inhibitors, and acute-phase proteins following anti-TNF-{alpha} therapy in rheumatoid arthritis. J. Immunol. 163: 1521-1528. [Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rossol, M.
Right arrow Articles by Wagner, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rossol, M.
Right arrow Articles by Wagner, U.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS