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The Journal of Immunology, 2007, 178, 7325 -7333
Copyright © 2007 by The American Association of Immunologists, Inc.
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Soluble TNF-Like Cytokine (TL1A) Production by Immune Complexes Stimulated Monocytes in Rheumatoid Arthritis1

Marco A. Cassatella2,*, Gabriela Pereira da Silva*, Ilaria Tinazzi{dagger}, Fabio Facchetti{ddagger}, Patrizia Scapini*, Federica Calzetti*, Nicola Tamassia*, Ping Wei§, Bernardetta Nardelli, Viktor Roschke||, Annunciata Vecchi#, Alberto Mantovani#, Lisa M. Bambara{dagger}, Steven W. Edwards** and Antonio Carletto{dagger}

* Department of Pathology, Division of General Pathology, and {dagger} Department of Clinical and Experimental Medicine, Division of Rheumatology, University of Verona, Verona, Italy; {ddagger} Department of Pathology, Spedali Civili, University of Brescia, Brescia, Italy; § Amgen, Thousand Oaks, CA 91301; Human Genome Sciences, Rockville, MD 20850; || CoGenesys, Rockville, MD 20850; # Fondazione Humanitas per la Ricerca, Rozzano, Italy; and ** School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TNF-like cytokine (TL1A) is a newly identified member of the TNF superfamily of ligands that is important for T cell costimulation and Th1 polarization. However, despite increasing information about its functions, very little is known about expression of TL1A in normal or pathological states. In this study, we report that mononuclear phagocytes appear to be a major source of TL1A in rheumatoid arthritis (RA), as revealed by their strong TL1A expression in either synovial fluids or synovial tissue of rheumatoid factor (RF)-seropositive RA patients, but not RF–/RA patients. Accordingly, in vitro experiments revealed that human monocytes express and release significant amounts of soluble TL1A when stimulated with insoluble immune complexes (IC), polyethylene glycol precipitates from the serum of RF+/RA patients, or with insoluble ICs purified from RA synovial fluids. Monocyte-derived soluble TL1A was biologically active as determined by its capacity to induce apoptosis of the human erythroleukemic cell line TF-1, as well as to cooperate with IL-12 and IL-18 in inducing the production of IFN-{gamma} by CD4+ T cells. Because RA is a chronic inflammatory disease with autoimmune etiology, in which ICs, autoantibodies (including RF), and various cytokines contribute to its pathology, our data suggest that TL1A could be involved in its pathogenesis and contribute to the severity of RA disease that is typical of RF+/RA patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Rheumatoid arthritis (RA)3 is a chronic idiopathic disease characterized by persistent inflammation of the synovium, leading to local destruction of bone and cartilage and a variety of systemic manifestations that lead to disability (1). RA involves all elements of the immune response, with autoimmune recognition of self-Ags by repeatedly activated autoreactive Th1 lymphocytes, the functional down-regulation and elimination of which appears defective (2). In addition, the local production of Igs and rheumatoid factors (RF), which are autoantibodies directed against the Fc portion of IgG, along with the local production of immune complexes (IC) leading to complement activation, appear important in the destructive events associated with the synovitis (3). Although the etiological stimulus has not been identified, a number of inflammatory mediators produced in the inflamed rheumatoid synovial tissue, including arachidonic acid metabolites, vasoactive amines, platelet-activating factor, proteinases, growth factors, and complement cleavage products, contribute to the inflammatory process (1). In addition, many of the local and systemic manifestations of RA appear to result from the production of a variety of cytokines within the inflamed synovium, particularly TNF-{alpha}, IL-1, IL-6, and IL-15, but also CD40/CD40L, B lymphocyte stimulator, receptor activator for NF-{kappa}B ligand, 41BB/41BBL, IL-18, chemokines, and angiogenic factors (4), contribute to the inflammatory process. Indeed, biological agents that specifically inhibit the effects of the cytokines implicated in the pathogenesis of RA (e.g., TNF-{alpha}, IL-1, or IL-15) can lead to a rapid improvement of inflammation, and substantial clinical benefits as compared with more conventional therapeutic approaches (5).

TNF-like cytokine (TL1A) is a newly identified member of the TNF superfamily of ligands, which has been identified as a longer variant of TL1 (also called VEGI) that binds the death domain receptor 3 (DR3; Refs. 6 and 7). Expression of DR3, a TNF superfamily receptor member with highest homology to TNFR1, is restricted to lymphocytes and is up-regulated upon T cell activation (8). TL1A can also bind to the soluble decoy receptor called TR6/DcR3, which also interacts with two other TNF ligands, namely Fas ligand and lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells, and competes with DR3 for TL1A binding (6). Like other TNF family ligands, TL1A contains a predicted transmembrane domain and a bioactive, proteolytically cleaved truncated form that can be released as a soluble factor (6). Recent studies have shown that recombinant soluble TL1A (sTL1A): augments IFN-{gamma} and GM-CSF production and increases IL-2 responsiveness by anti-CD3/CD28-stimulated T cells (6); synergizes with IL-12 and IL-18 to augment IFN-{gamma} release in human T and NK cells (8); and biases T cells to differentiate toward the Th1 phenotype (9, 10). It has also been shown that resting T cells, B cells, NK cells, dendritic cells, and monocytes do not express TL1A mRNA, in contrast with HUVECs stimulated by TNF, IL-1{alpha}, or PMA (6) or CD11chigh dendritic cells in mice with inflammation (10). In contrast, membrane-bound TL1A (mTL1A) was reported as expressed by in vitro activated T cells and by a subset of mucosal T cells and macrophages in inflamed intestinal mucosa in Crohn’s disease and ulcerative colitis (9).

In this work, we explored whether activated human leukocytes can produce sTL1A and, if so, which leukocyte subtype was responsible. We show that mononuclear phagocytes are potent producers of sTL1A and that insoluble IC are extremely strong activators of this secretion. These observations led us to investigate whether TL1A is expressed in pathological conditions associated with increased levels of circulating or deposited IC, such as RA. Remarkably, we found a marked expression of TL1A associated with mononuclear phagocytes present in synovial membranes and synovial fluids (SF) of medication-free RF+/RA patients at first diagnosis with active disease. Our data imply that, in vivo, monocytes and macrophages of RA patients express TL1A in response to IC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell purification and culture

Leukocytes were isolated under endotoxin-free conditions from either buffy coats of healthy donors or whole blood of healthy donors and patients, by standard procedures. Briefly, buffy coats or blood were layered on Ficoll-Hypaque density gradient (Amersham Biosciences) and then centrifuged at 400 x g for 30 min at room temperature. The interface between plasma and Ficoll, corresponding to the mononuclear cell (PBMC) fraction, was then collected, washed five times with PBS to eliminate platelets, suspended in isosmotic (285 mOsm) culture medium (RPMI 1640 containing 10% low endotoxin FCS and 4 mM HEPES), and layered over a 46% isosmotic Percoll solution (Pharmacia). After centrifugation (30 min at room temperature, 650 x g), the monocyte and lymphocyte fractions were collected, washed in PBS, and suspended in standard culture medium (as described later in this paragraph). Monocyte preparations were >85% pure as evaluated by both CD14 expression by FACS and morphology after May-Grünwald-Giemsa staining of cytospins. In some experiments, monocytes were also isolated by negative selection using MACS separation (Monocyte Isolation kit II; Miltenyi Biotec) to >95% purity. Neutrophils were isolated by Ficoll centrifugation followed by dextran sedimentation and osmotic lysis and were >95% pure (as evaluated by May-Grünwald-Giemsa staining) (11). After purification, cells were suspended in RPMI 1640 supplemented with 10% low-endotoxin FCS (<0.5 endotoxin U/ml; BioWhittaker; defined as standard culture medium), plated in six 24-well tissue culture plates (Orange Scientific Cambrex, Techno Plastic Products) at 5 x 106/ml, and then cultured for up to 48 h in a 5% CO2 atmosphere in the presence or absence of various stimuli, including 100 ng/ml LPS (from Escherichia coli, serotype 026:B6; Sigma-Aldrich), 5 ng/ml TNF-{alpha}, 10 ng/ml GM-CSF, 10 ng/ml IL-4, 10 ng/ml IL-13 (Peprotech), 200 U/ml IFN-{gamma} (R&D Systems), 1000 U/ml IFN{alpha} (Roferon; Roche Laboratories), 200 U/ml IL-10 (DNAX and Schering-Plough), 100 nM fMLP (Sigma-Aldrich), and 50 µg/ml insoluble immune complexes (IC). The latter were prepared by using OVA from chicken egg (Sigma-Aldrich) or BSA (Sigma-Aldrich) as Ags, with, respectively, anti-albumin chicken egg rabbit antiserum (Calbiochem) and anti-BSA rabbit antiserum (Calbiochem) as previously described (12). Endotoxin contamination of IC (at the working concentrations) was <0.06 endotoxin U/ml (corresponding to ~6 pg/ml), as determined by the Limulus amebocyte lysate assay (BioWhittaker). For direct Fc{gamma}R engagement, 96- or 24-well plates were coated with purified human IgG (Sigma-Aldrich) at 20 µg/0.2 ml/well in sterile PBS for 2 h and washed three times with warm PBS before use (13). In selected experiments, before stimulation, monocytes were preincubated with: 20 µg/ml polymyxin B sulfate (PMX); anti-TNF-{alpha}-neutralizing mAbs (B154.2 clone; Ref. 14); 10 ng/ml IL-4; 200 U/ml IFN-{gamma}. Alternatively, monocytes were preincubated for 1 h at 4°C with 10 µg/ml anti-Fc{gamma}R Abs and related isotype controls (IgG1 for all of them) in the absence of FCS. Anti-Fc{gamma}R Abs were: anti-Fc{gamma}RI/CD64 (clone 10.1; Ancell); anti-Fc{gamma}RII/CD32 (AT10 clone, which binds to all three forms of Fc{gamma}RII (a, b, and c); Serotec) and anti-Fc{gamma}RIII/CD16 (3G8 clone; BD Pharmingen). IgG1 were purchased from BD Pharmingen. Monocytes were then washed, suspended in medium, plated, and then stimulated. After culture, cells were detached, collected, and spun at 350 x g for 5 min; the resulting supernatants were immediately frozen in liquid nitrogen and stored at –80°C. All reagents were of the highest available grade and were dissolved in pyrogen-free water suitable for clinical use.

Patients

RA patients, selected according to the American Rheumatism Association criteria (15), were enrolled at first diagnosis after their signed informed consent and approval by the local ethical committees. These patients were categorized as having early, disease-modifying anti-rheumatic drug-free, active-phase disease (with disease activity score of >3.5) and grouped into RF-seropositive (RF+/RA) or RF-seronegative (RF–/RA), according to the detection of threshold levels of both serum IgM RF (≥10 U/ml, as measured by nephelometry) and IgG RF (≥6 U/ml, as measured by QUANTA Lite RF IgG ELISA (Menarini Diagnostics). Age- and sex-matched healthy subjects were enrolled as controls.

SF samples

SF samples were collected from RF+/RA and RF–/RA patients, and, as control, osteoarthritis (OA) patients who underwent therapeutic and/or diagnostic arthrocentesis of a knee effusion. After withdrawal, samples were retained for cytospins and subsequent immunocytochemistry, whereas cell-free SF supernatants were isolated for subsequent analyses.

Polyethylene glycol (PEG) precipitation of serum ICs

Sera from RF+/RA, RF–/RA, and healthy donors were precipitated with 3% (w/v) ice-cold PEG 6000 (Sigma-Aldrich), centrifuged, washed three times in sterile PBS, and finally diluted to the initial serum volume in sterile PBS as described (16). Cell-free SF (from RF+/RA patients) was centrifuged at 11,600 x g for 5 min to pellet insoluble ICs (which were further purified by PEG precipitation as above), whereas the soluble IC retained in the supernatant was discarded (17).

Analysis of mediator concentration

sTL1A concentrations in cell-free supernatants and freshly prepared sera and SF were measured by a specific ELISA developed at Human Genome Sciences (Rockville, MD; detection limit, 20–40 pg/ml). Briefly, flat-bottom 96-well plates (MaxiSorp 439454; Nunc) were coated with 75 µl/well of 2 µg/ml anti-TL1A capture mAb (clone 04H08), diluted in PBS (pH 7.6), and incubated overnight at 4°C. After an extensive rinsing in washing buffer (0.05% Tween 20 in PBS, pH 7.2–7.4), the plates were incubated for at least 1 h at room temperature with 225 µl/well blocking buffer (1% BSA, 5% sucrose in PBS). After a washing, 75 µl of either recombinant TL1A standards or samples were added to the wells and incubated for 2 h at room temperature. Plates were then extensively washed before addition of 75 µl/well detection biotin-conjugated mAb (clone 16H02) suspended in reagent diluent (1% BSA in PBS, pH 7.2–7.4, 0.2 µm filtered) and incubated for 2 h at room temperature. After extensive washings, 75 µl/well of streptavidin-HRP (SNN2004; Biosource International) diluted 1/10,000 in reagent diluent were added, and plates were incubated for 45–60 min at room temperature in the dark. After a rinsing, 75 µl/well substrate solution containing 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) were added and incubated for 20 min at room T in the dark. The reaction was stopped with 50 µl/well of 2 N H2SO4 and optical densities were measured at 450 nm using a microplate reader (Packard Spectracount BSL0001). All assays were conducted in duplicate. A second TL1A ELISA purchased from Peprotech (detection limit, 62 pg/ml) was used according to the manufacturer’s instructions, which fully reproduced the data obtained with the Human Genome Sciences ELISA. IL-1 receptor antagonist (IL-1ra) and TNF-{alpha} were measured by using ELISA kits purchased from R&D Systems, whereas IFN-{gamma} was determined by the ELISA kit obtained from ImmunoTools.

Northern blotting and real-time RT-PCR analysis

These experiments were conducted as described previously (6, 11). For real-time RT-PCR, beta2-microglobulin was selected as a normalizing gene, due to its stable expression levels in leukocytes (18). Data were calculated with Q-Gene software (www.BioTechniques.com) and are expressed as mean normalized expression units after beta2-microglobulin normalization.

Flow cytometry for mTL1A expression

Freshly isolated or activated monocytes were stained with mAbs raised against human TL1A (12F11; Human Genome Sciences) or control mouse IgG1 (Sigma-Aldrich), followed by biotinylated secondary sheep anti-mouse IgG (Sigma-Aldrich). Alternatively, TL1A staining was performed with polyclonal biotinylated Abs from R&D (BAF744) or goat biotinylated IgG (BD Pharmingen) as isotype controls. CR3 staining was performed with OKM1 mAb (Caltag Laboratories). Immunolabeling was achieved after the addition of PE-conjugated streptavidin (BD Biosciences). Cytofluorographic analyses (using at least 104 cells/sample) were performed on a FACScan (BD Biosciences) using CellQuest software (19).

Apoptosis of TF-1 cells

The biological activity of sTL1A released by activated monocytes into culture medium was tested by the ability of monocyte-derived supernatants to induce apoptosis of the human erythroleukemic cell line, TF-1, as recently described (6). Briefly, TF-1 cells (8 x 105 cells/ml) were suspended either directly in supernatants harvested from resting or IC-stimulated monocytes, or, as control, in standard culture medium containing various concentrations of recombinant TL1A and then seeded in 96-well culture plates (Orange Scientific Cambrex). Cell cultures were incubated in the presence or absence of 10 µg/ml cycloheximide (CHX), as described (6). After 6 h, TF-1 cells were harvested, and their apoptosis was measured with an Annexin V Fluos staining kit (Roche; Ref. 20). To assess the specificity of the TL1A-mediated proapoptotic activity, monocyte-derived supernatants and recombinant human TL1A were preincubated with 600 ng/ml anti-TL1A mAbs (clone 16H02) or isotype-matched control mAbs (IgG1) for 20 min at 37°C. All monocyte-derived supernatants used for these experiments were previously immunodepleted of TNF-{alpha} using well-established procedures (21, 22). Complete removal of TNF-{alpha} was verified by ELISA quantification of TNF-{alpha}-immunodepleted supernatants.

IFN-{gamma} production assay

Human CD4+ T lymphocytes were isolated from the Percoll lymphocyte fraction using a negative selection enrichment kit for human CD4+ T cells (StemCell Technologies), according to the manufacturer’s instructions. Purity was routinely >97%, as determined by FACS. CD4+ T cells were cultured at 106/ml in the presence or absence of 2 ng/ml IL-12 (PeproTech) plus 50 ng/ml IL-18 (R&D Systems), either singly or in combination with various concentrations of recombinant TL1A. CD4+ T cells were also cultured with or without IL-12 plus IL-18 in supernatants harvested from resting or IC-stimulated monocytes (previously immunodepleted of TNF-{alpha}, as described in "Apoptosis of TF-1 cells"). After 72 h of incubation, supernatants were collected and assayed for IFN-{gamma} content by ELISA. To assess the specific effect of the TL1A contained in monocyte-derived supernatants, the latter were preincubated with 1 µg/ml anti-TL1A or isotype-matched control mAbs for 20 min at 37°C.

TL1A expression in RA tissue

Synovial sections explanted from six RF+/RA patients, two RF–/RA patients, and, as controls, from four patients with synovitis associated with chronic OA were examined by immunohistochemistry. In addition, s.c. nodules from patients with active RA displaying typical rheumatoid granulomas were analyzed. All specimens were formalin fixed and paraffin embedded. Serial sections were stained with mAbs against TL1A (clone 12F11; overnight incubation at 1/3000), CD3 (1/50), CD20 (1/200) and CD68 (1/50) (all from DakoCytomation), followed by a peroxidase-conjugated dextran polymer (ChemMate; DakoCytomation). Sections were developed using diaminobenzidine as chromogen and Mayer’s hematoxylin as counterstaining. Staining for TL1A on cells obtained upon cytocentrifugation of SFs from two RA patients followed the same procedure. For double immunofluorescence for CD14 and TL1A, sections were first incubated with anti-CD14 (clone MM42; IgG2a; Novo Castra; 1/50), followed by Texas red-conjugated anti-IgG2a (SouthernBiotech), anti-TL1A, and FITC-conjugated anti-IgG1 (SouthernBiotech). Negative controls consisted of the omission of primary Abs and by the use of irrelevant isotype matched Abs. Preincubation of 12F11 mAbs with IgM-RF or IgG-RF preparations (508692 and 708685, respectively, from Inova-San Diego) before their addition to tissue specimens did not modify the outcome of the final TL1A staining. Sections were then finally examined with an Olympus BX60 microscope and equipped with relevant fluorescence excitation/emission filters and a DP-70 digital camera (Olympus). Images were acquired using analySIS ImageProcessing software (Soft Imaging System); immunofluorescence images were merged as previously described (23).

Statistical analysis

Data are expressed as means ± SEM. Statistical evaluation was performed by Student’s t test for paired data and considered to be significant if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Human monocytes incubated with insoluble IC release sTL1A

To investigate whether mononuclear phagocytes express TL1A, human monocytes isolated from buffy coats of healthy donors were cultured for up to 48 h with optimal concentrations of various inflammatory mediators, including LPS (n = 13), TNF-{alpha} (n = 6), GM-CSF (n = 6), fMLP (n = 6), IFN-{gamma} (n = 6), IFN{alpha} (n = 3), IL-4 (n = 6), IL-10 (n = 3), IL-13 (n = 3), aggregated IgG (n = 4), and BSA/anti-BSA or OVA/anti-OVA insoluble IC (n = 13). Cell-free supernatants were collected to determine the yields of sTL1A, whereas adherent cells were processed for TL1A mRNA accumulation by Northern blot analysis and/or real time RT-PCR. Of all these stimuli, only insoluble IC and aggregated IgG stimulated a remarkable extracellular release of sTL1A by 20-h-cultured monocytes (596 ± 83 pg/ml for OVA/anti-OVA IC, n = 10; 683 ± 152 pg/ml for BSA/anti-BSA IC, n = 15; 258.4 ± 62.1 pg/ml, n = 10, for aggregated IgG). This effect of insoluble IC toward sTL1A release was selective in that their capacity to elicit the production of IL-1ra was substantially similar to that triggered by LPS, which was only a modest activator of sTL1A release (61.2 ± 15.5 pg/ml; n = 15) compared with untreated monocytes (<20 pg/ml, n = 15). Northern blot experiments revealed that sTL1A production by insoluble IC-activated monocytes is preceded by an up-regulation of TL1A mRNA expression, which progressively increased over time and reached maximal expression between 6 and 20 h of incubation, depending on the donor (Fig. 1A). In contrast, up-regulation of TNF-{alpha} and IL-1ra mRNA expression in insoluble IC-treated monocytes occurred with faster kinetics (Fig. 1, A and B). Similar results were obtained by real-time RT-PCR studies, which also confirmed the very weak up-regulatory effect of LPS on TL1A mRNA expression compared with that induced by OVA/anti-OVA insoluble IC (Fig. 1B) and BSA/anti-BSA insoluble IC (data not shown). Time-course studies demonstrated that the ability of insoluble IC and LPS to stimulate the release of sTL1A from monocytes is relatively slow (starting 5 h poststimulation and progressively increasing up to 48 h of incubation) (Figs. 1C and 2A), being more delayed compared with the kinetics of TNF-{alpha} (Fig. 1C) or IL-1ra release (data not shown). In addition, dose-response experiments revealed that concentrations as low as 2.5–5 µg/ml and 1–10 ng/ml insoluble IC and LPS, respectively, can trigger a detectable sTL1A production (Fig. 2B). Preincubation of OVA/anti-OVA or BSA/anti-BSA insoluble IC with PMX did not suppress their capacity to induce the production and secretion of sTL1A or IL-1ra, whereas it completely blocked the effect of LPS (Fig. 2C), thus excluding endotoxin contamination of our IC preparations (see also Materials and Methods for Limulus amebocyte lysate assay results). Surprisingly, only activated monocytes, but neither neutrophils nor lymphocytes, were able to release sTL1A under these experimental conditions (Fig. 2D). Activated neutrophils, however, did produce considerable amounts of IL-1ra (data not shown), in line with previous results (24). Experiments in which we added anti-TNF-{alpha}- neutralizing Abs to insoluble IC monocytes indicated that monocyte-derived TNF-{alpha} does not play a significant role in inducing TL1A (data not shown). All these results have been reproduced with a second, commercially available TL1A ELISA (see Materials and Methods).


Figure 1
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  		FIGURE 1. TL1A mRNA expression in IC-stimulated monocytes. Purified populations of monocytes were incubated for the times indicated with 50 µg/ml BSA/anti-BSA insoluble IC (A) or 50 µg/ml OVA/anti-OVA insoluble IC or 100 ng/ml LPS (B). TL1A, TNF-{alpha}, and actin mRNA expression was then detected either by Northern blotting analysis (A) or by real-time RT-PCR (B). B, Gene expression is depicted as mean normalized expression (MNE) units after beta2-microglobulin normalization of triplicate reactions for each sample. Data are representative of results from three independent experiments for each panel. C, Determination of sTL1A and TNF-{alpha} levels in supernatants harvested from the experiment shown in A.

 

Figure 2
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  		FIGURE 2. Characterization of TL1A production by activated monocytes. A and B, Monocytes (5 x 106/ml) were incubated either for various times with 50 µg/ml OVA/anti-OVA insoluble IC or 100 ng/ml LPS (A) or with increasing concentrations of stimuli for 20 h (B), before determination of sTL1A levels in cell-free supernatants. C, Monocytes were preincubated for 30 min with 20 µg/ml PMX before stimulation with OVA/anti-OVA insoluble IC or 100 ng/ml LPS. D, PBMC, monocytes, lymphocytes, and neutrophils purified from the same donor were cultured at 5 x 106/ml for 20 h with or without 50 µg/ml OVA/anti-OVA insoluble IC and 100 ng/ml LPS. Culture supernatants were then collected for sTL1A and IL-1ra measurement. Values are means ± SEM of duplicate determinations calculated from three to four independent experiments.

 
Finally, and in contrast with the remarkable release of sTL1A, expression of mTL1A increased only slightly in insoluble IC-treated monocytes, as assessed by flow cytometry using Abs from two different sources (Fig. 3). Collectively, our data unequivocally identify insoluble IC as very potent and specific stimuli for the induction of both TL1A mRNA expression and sTL1A release in human monocytes, but not in neutrophils or lymphocytes.


Figure 3
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  		FIGURE 3. TL1A surface expression in activated monocytes. Monocytes were cultured for 20 h with or without 50 µg/ml OVA/anti-OVA insoluble IC and 100 ng/ml LPS before analysis of membrane-bound TL1A (mTL1A) and CR3 expression by flow cytometry. Isotype anti-mouse IgG1 mAbs were used as controls. Data are representative of results from five independent experiments.

 
Biological activities of monocyte-derived sTL1A

To ascertain whether sTL1A released by IC-stimulated monocytes was biologically active, we tested the ability of monocyte-derived supernatants to induce the apoptosis of the TL1A-sensitive TF-1 cells (6). As shown by annexin V staining (Fig. 4A), sTL1A that accumulated in conditioned medium obtained from IC-stimulated monocytes was able to potentiate the proapoptotic effect of CHX on TF-1 cells by ~2-fold. These supernatants contained ~0.5–1 ng/ml sTL1A, and at these concentrations recombinant TL1A induced identical effects of apoptosis on CHX-treated TF-1 cells (Fig. 4A). No significant apoptosis of TF-1 cells was observed if TL1A was used in the absence of CHX, or if TF-1 cells were incubated in medium from resting monocytes (Fig. 4A). Importantly, promotion of TF-1 cell apoptosis by conditioned medium obtained from IC-stimulated monocytes was TL1A-specific because it was completely abrogated by neutralizing anti-TL1A mAbs (Fig. 4A), but not by isotype-matched control Abs (data not shown). All monocyte-derived supernatants used in these assays were subjected to TNF-{alpha} removal by immunoabsorption (21, 22). This was necessary because preliminary experiments revealed that recombinant TNF-{alpha}, used at the same concentrations as those detected in conditioned medium harvested from IC-activated monocytes (ranging from 5 to 25 ng/ml, depending on the donor), also exerted potent proapoptotic activity on CHX-treated TF-1 cells.


Figure 4
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  		FIGURE 4. Supernatants from IC-stimulated monocytes promote apoptosis of TL1A-sensitive cells and IFN-{gamma} production by CD4+ T cells stimulated with IL-12 plus IL-18. A, TF-1 cells were suspended in supernatants harvested from resting or IC-stimulated monocytes that were previously immunodepleted of TNF-{alpha} (see Materials and Methods) and, as control, in standard culture medium containing 0.5 ng/ml recombinant TL1A and then cultured in the presence or the absence of 10 µg/ml CHX. Aliquots of monocyte-derived supernatants or recombinant TL1A were also preincubated with 600 ng/ml neutralizing anti-TL1A mAbs or isotype-matched control mAbs for 30 min to assess the specificity of the TL1A-mediated proapoptotic activities. After 6 h, TF-1 cells were collected, and apoptosis was assessed by annexin V staining by flow cytometry analysis. Means ± SEM of the relative percent of annexin V-positive cells calculated from three independent experiments are shown. B, CD4+ T cells were incubated at 106/ml in standard culture medium with or without 2 ng/ml IL-12 plus 50 ng/ml IL-18, singly or in combination with either supernatants harvested from resting or IC-stimulated monocytes (previously immunodepleted of TNF-{alpha}), or 1 ng/ml recombinant TL1A. After 72 h of incubation, supernatants were collected and assayed for IFN-{gamma} content by ELISA. Aliquots of monocyte-derived supernatants or recombinant TL1A were also preincubated with 1 µg/ml neutralizing anti-TL1A mAbs or isotype matched control mAbs for 30 min. Means ± SEM of IFN-{gamma} release calculated from three independent experiments are shown. *, Significant effects; *, p < 0.05; **, p < 0.01.

 
Subsequently, we examined whether monocyte-derived supernatants could augment the production of IFN-{gamma} by IL-12 plus IL-18-stimulated CD4+ T cells, as described by Papadakis et al. (8). As shown in Fig. 4B, sTL1A that accumulated in medium harvested from IC-stimulated monocytes was able to significantly potentiate the ability of IL-12 plus IL-18 to trigger CD4+ T cell production of IFN-{gamma} by ~8-fold (n = 3). These monocyte-derived supernatants were previously immunodepleted of TNF-{alpha} (as described previously) and contained ~1 ng/ml sTL1A, as determined by ELISA. Recombinant TL1A at 1 ng/ml used together with IL-12/IL-18 was less potent than monocyte-derived supernatants (Fig. 4B), suggesting that other factor(s) present in supernatants of IC-stimulated monocytes synergistically cooperate with endogenous sTL1A in inducing the production of IFN-{gamma}. No significant effects were observed if TL1A was used in the absence of IL-12/IL-18 or if CD4+ T cells were incubated in medium from resting monocytes (Fig. 4B). Importantly, stimulation of IFN-{gamma} production by conditioned medium obtained from IC-stimulated monocytes was TL1A specific because it was completely abrogated by neutralizing anti-TL1A mAbs (Fig. 4B), but not by isotype-matched control Abs (data not shown).

Effect of Fc{gamma}R blocking and immunoregulatory cytokines on the ability of insoluble IC to trigger sTL1A production by monocytes

To establish which Fc{gamma}R(s) was responsible for triggering TL1A expression, monocytes were pretreated for 1 h with anti-Fc{gamma}RI/CD64, anti-Fc{gamma}RII/CD32, anti-Fc{gamma}RIII/CD16, and related isotype control Abs before their stimulation with BSA/anti-BSA insoluble IC for 20 h. As shown in Fig. 5A, production of sTL1A in IC-stimulated monocytes was reduced by 65 ± 13% and 20 ± 5%, respectively, by anti-Fc{gamma}RII/CD32 and anti-Fc{gamma}RIII/CD16 Abs but was not affected by anti-Fc{gamma}RI/CD64 (Fig. 5A) or by isotype control Abs (data not shown). Anti-Fc{gamma}R Abs alone did not trigger, by themselves, any sTL1A expression (data not shown). These data suggest that CD32 appears to be the major Fc{gamma}R required for the induction of sTL1A by insoluble IC in human monocytes.


Figure 5
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  		FIGURE 5. Effect of Fc{gamma}R blocking and immunoregulatory cytokines on the ability of insoluble IC to trigger sTL1A production by monocytes. A, Monocytes were preincubated with 10 µg/ml anti-Fc{gamma}RI/CD64, anti-Fc{gamma}RII/CD32, and anti-Fc{gamma}RIII/CD16 and related isotype control Abs in the absence of FCS for 1 h at 4°C. Cells were then washed, suspended in complete medium, plated, and then incubated with 50 µg/ml BSA/anti-BSA insoluble IC. After 20 h, culture supernatants were collected for sTL1A measurement and analysis. Bars depict the mean values ± SEM (n = 3) of the percentage of inhibition exerted by the various anti-Fc{gamma}R Abs on the production of sTL1A triggered by insoluble IC. B and C, Monocytes were preincubated with 10 ng/ml IL-4 (B) or 200 U/ml IFN-{gamma} (C) for 1 h before stimulation with 50 µg/ml BSA/anti-BSA insoluble IC, 50 µg/ml OVA/anti-OVA insoluble IC, or 100 ng/ml LPS. After 20 h, culture supernatants were collected for sTL1A measurement and analysis. Bars depict the mean values ± SEM (n = 4) of the percentage of inhibition or fold increase exerted by IL-4 or IFN-{gamma}, respectively.

 
We also investigated whether IL-4 and IFN-{gamma} might influence the inducible expression of TL1A. Fig. 5B shows that IL-4 almost completely abolished monocyte production of TL1A induced by LPS, OVA/anti-OVA, and BSA/anti-BSA insoluble IC. In contrast, IFN-{gamma} greatly potentiated the stimulatory effect of all these agonists, particularly LPS and BSA/anti-BSA insoluble IC (Fig. 5C). Consistent with these data, real-time RT-PCR experiments mirrored the modulatory actions of both IL-4 and IFN-{gamma} at the level of TL1A mRNA accumulation (data not shown).

TL1A detection in synovial tissue of RA patients

The identification of insoluble IC or aggregated IgG as potent stimuli for sTL1A production in vitro led us to hypothesize that sTL1A could be expressed in RA patients. Ig-containing IC or autoantibodies such as RF are abundant in serum and synovial fluid of RA patients (25, 26) and predict a more aggressive, destructive course of disease (1). Because we could not accurately measure the levels of sTL1A in serum or synovial fluid samples due to the interference of RF in our two TL1A ELISAs, we performed an immunohistochemical analysis of both synovial and skin tissue samples and SF cells from RA patients. Fig. 6A shows that synovium from RF+ patients with active RA contained high numbers of cells that strongly expressed TL1A in their cytoplasm (Fig. 6A, b and c), whereas synovium from OA patients showed only few TL1A-positive (TL1A+) cells (Fig. 6Aa). These TL1A+ cells in RA patients displayed a monocyte-macrophage morphology and were dispersed among other inflammatory cells, being particularly abundant beneath the synovium. Remarkably, in rheumatoid nodules of the subcutaneous tissue, the TL1A+ cells formed the palisade surrounding the central area of fibrinoid necrosis (Fig. 6Ae). The mononuclear phagocyte origin of the TL1A+ cells was supported by the staining of serial sections with anti-CD68 (Fig. 6Ac, anti-TL1A; Fig. 6Ad, anti-CD68). This was confirmed by double immunofluorescence with TL1A and anti-CD14 (Fig. 6Af). Similar to the study of Bamias et al. (9), a few TL1A-expressing plasma cells were occasionally found (data not shown), whereas no TL1A+ lymphocytes, polymorphonuclear neutrophils, or endothelial cells were observed (Fig. 6Ac). In areas of marked inflammation adjacent to the synovial surface, TL1A positivity appeared to be associated with the synoviocytes, possibly due to release of the cytokine from closely associated macrophages (Fig. 6Ab). Interestingly, TL1A staining of synovial samples from RF–/RA patients was much less intense and characterized by paucicellular infiltrate, other than a TL1A-negative synovium (data not shown). Finally, mononuclear cells with monocyte/macrophage morphology present in the SF of RF+/RA patients were intensely stained by the anti-TL1A Ab; the staining was confined to the cytoplasm and not detected at the membrane (Fig. 6B).


Figure 6
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  		FIGURE 6. TL1A expression in synovial tissue and synovial exudates of RA patients. A, TL1A expression in synovial tissue from a patient with osteoarthritis (a, upper left), and in synovial tissue (b–d and f) and skin (e) from patients with RA (b–f). Whereas in OA synovial tissue only few stromal macrophages are TL1A positive, in RA many positive cells are found both in the inflamed stroma, and near the surface of the tissue (b). Serial sections stained with anti-TL1A (c) and anti-CD68 (d) show similar distributions of the positive cells, indicating that the TL1A+ cells are macrophages. Note the negativity of the lymphocytes within the nodule, as well as of endothelium. Macrophages forming the palisade in a typical rheumatoid granuloma are strongly TL1A+ (e). Double immunofluorescence for CD14 (red) and TL1A (green) reveals a colocalization of the two Ags in the vast majority of cells (f). B, Cytospins were prepared by spinning 15 x 105 SF cells per spot in a Shandon Cytospin 3 centrifuge. Mononuclear cells within the synovial fluid show cytoplasmic TL1A staining, but the neutrophils within this sample fail to stain.

 
PEG precipitates from serum or insoluble IC purified from SF of RF+/RA patients induce the release of sTL1A by monocytes

The above findings lead us to speculate that serum or SF IC from RA patients might induce circulating/inflammatory monocytes or synovial macrophages to produce and secrete sTL1A. To verify this hypothesis, monocytes isolated from healthy subjects were cultured for 20 h in standard medium containing different dilutions of PEG precipitates prepared from the serum of RF+/RA patients, RF–/RA patients, or healthy subjects. As shown in Fig. 7A, only PEG precipitates prepared from the serum of RF+/RA patients were able to stimulate, dose dependently, the release of very high amounts of sTL1A by monocytes, whereas the control RF–/RA precipitates did not. Similarly, insoluble IC purified from the SF of two RF+/RA patients also potently stimulated sTL1A release by monocytes (Fig. 7B). These insoluble SF IC were shown also to be potent activators of the neutrophil respiratory burst (data not shown), as previously described (12). Collectively, these data are consistent with the notion that, in RF+/RA patients, the production of sTL1A by mononuclear phagocytes is triggered by IC present in biological fluids (including SF).


Figure 7
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  		FIGURE 7. PEG precipitates from RF+/RA sera and SFs induce the production of sTL1A by monocytes. A, Serum samples (250 µl) from RF+/RA patients and healthy donors were subjected to PEG precipitation and then suspended in 250 µl of PBS. Dilutions (1/25, 1/10 and 1/5 v/v, in standard medium) of each precipitate were added to cultures of monocytes isolated from healthy donors. Similarly, insoluble IC purified from SF of two RF+/RA patients were added in a 250-µl volume to cultures of monocytes isolated from healthy donors (B). After 20 h, monocyte-conditioned medium were harvested and centrifuged, and the resulting supernatants were subjected to sTL1A determination. Optical densities of the PEG precipitates were also determined and, if found above the blanks, they were subtracted from the optical densities of the corresponding monocyte-derived conditioned medium. Bars show the net values of sTL1A production by monocytes. Values show means of duplicate determinations calculated from one experiment representative of two for A and from the two SF patients in B.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TL1A (TNFSF15/VEGI) is a recently identified member of the TNF superfamily that interacts with DR3, costimulates T cells augmenting anti-CD3 plus anti-CD28 IFN-{gamma} and GM-CSF production, and, importantly, biases T cells to differentiate toward the Th1 phenotype (6, 8, 9, 27). However, although much is becoming known about TL1A function, very little is known about its expression in normal or pathological states.

In this study, we have demonstrated that, of the various leukocytes examined, only monocytes can be induced to accumulate TL1A mRNA and release significant quantities of sTL1A in response to physiological concentrations (28, 29) of insoluble ICs. sTL1A released into medium by insoluble IC-stimulated monocytes was biologically functional because it specifically synergized with CHX in inducing the apoptosis of the TF-1 cells (6) and also cooperated with IL-12 plus IL-18 in inducing the production of IFN-{gamma} by CD4+ T cells (8). Although LPS also stimulated the release of monocyte-derived sTL1A, its effect was very weak compared with that of insoluble IC. Release of sTL1A by monocytes was slow, delayed in comparison with other inflammatory cytokines (such as TNF-{alpha}), but was sustained over time and dependent on the concentration of the stimuli. The selective action of insoluble IC on sTL1A production was emphasized by the fact that a variety of classical mediators, including TNF-{alpha}, GM-CSF, fMLP, CXCL8, IFN-{gamma}, IFN{alpha}, IL-4, IL-10, and IL-13, all failed to up-regulate TL1A gene and protein expression. PBLs and neutrophils cultured with insoluble IC, LPS, or other proinflammatory stimuli were completely unable to release sTL1A or to express mTL1A (P. Scapini, F. Calzetti, and M. A. Cassatella, unpublished observations), further highlighting that monocytes are the major cellular source and insoluble IC the selective stimuli for sTL1A production. In contrast, expression of mTL1A in insoluble IC-stimulated monocytes was minimal, in line with the observations made in LPS-activated monocytes (30). Finally, we have also shown that the molecular interaction between BSA/anti-BSA insoluble IC and monocytes is mainly via Fc{gamma}RII/CD32 (rather than CD64 or CD16), at least under our experimental conditions. Previous finding reporting that Fc{gamma}RIII{alpha}/CD16 plays a key role in mediating the induction of both TNF-{alpha} and IL-1{alpha} production by human macrophages of RA patients following receptor ligation by small ICs (31) highlight the fact that the mechanisms governing the expression of TL1A and TNF-{alpha}/IL-1{alpha} might be subjected to distinct regulatory pathways or depend on the cell type.

Our in vitro observations prompted us to hypothesize whether sTL1A expression occurred in vivo in conditions such as RA, in which immune complexes and/or autoantibodies are elevated. In support of this hypothesis, we have demonstrated that monocytes incubated either with PEG precipitates prepared from the serum of RF+/RA patients, which are enriched for IC (32), or with insoluble IC purified from the SF of RF+/RA patients potently stimulated monocytes to release high amounts of sTL1A in vitro. Consistent with a role for monocytes as a TL1A source in RA, synovial tissue samples from active RA patients were characterized by a strong expression of TL1A in macrophages, in keeping with the results of Bamias et al. (9), who also found that the main source of TL1A in inflammatory bowel disease are monocyte-derived cells. Furthermore, synovial monocytes present in the SF of RF+/RA patients were intensely TL1A positive. Our data strongly favor the notion that, in RF+/RA patients, IC stimulate mononuclear phagocytes to produce and release sTL1A, either into the blood or locally, for example, into inflamed joints. We observed that the capacity of PBMC from RA patients to produce sTL1A when stimulated with insoluble IC in vitro was identical with that of normal PBMC (M. A. Cassatella, G. Pereira da Silva, I. Tinazzi, F. Calzetti, unpublished observations).

Although the functional significance of sTL1A in RA remains to be clarified, one possibility is that TL1A contributes to sustain the inflammatory process. For example, in monocytic cell lines, recombinant TL1A used in association with IFN-{gamma} induces both CXCL8 and matrix metalloproteinase-9 production (30), two mediators implicated in local cell infiltration and joint degradation in RA patients (33, 34). Alternatively, given the ability of TL1A to modulate T lymphocyte functions, as demonstrated by its capacity to maintain mucosal Th1 polarization in patients with Crohn’s disease (9) or in two models of chronic murine ileitis (10), it is possible that the TL1A production by IC-stimulated monocytes might also be involved in directing and sustaining the various Th1-driven immunological responses in RA. In support of this notion, our experiments indicated that IL-4 and IFN-{gamma} suppress and enhanc, respectively, the production of sTL1A by insoluble IC-treated monocytes, which is exactly what one would expect if TL1A is regulated by the classical Th1-type cytokines.

In conclusion, it is well known that there is a short window of opportunity in the early onset of RA, which, if identified and treated in a timely manner, could lead to a better prognosis (35). Therefore, understanding the early events and identifying the precise markers that predict aggressive synovitis would aid in the design of therapeutic strategies. Our data might have identified sTL1A as a novel therapeutic target.


   Acknowledgment
 
We thank Francesca Gentili for her excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials 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 This work was supported by grants (to M.A.C.) from Ministero dell’Istruzione, dell’Università e della Ricerca (Progetti di Rilevante Interesse Nazionale 2005, Fondo di Investimento per la Ricerca di Base, and 60%), Fondazione Cassa di Risparmio, and Associazione Italiana per la Ricerca sul Cancro. Back

2 Address correspondence and reprint requests to Dr. Marco A. Cassatella, Division of General Pathology, Department of Pathology, Strada Le Grazie 4, Verona, Italy. E-mail address: marco.cassatella{at}univr.it Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; IC, immune complex; RF, rheumatoid factor; SF, synovial fluid; TL1A, TNF-like cytokine; sTL1A, soluble TL1A; mTL1A, membrane-bound TL1A; DR3, death domain receptor 3; IL-1ra, IL-1 receptor antagonist; PMX, polymyxin B sulfate; RF+/RA, RF-seropositive RA patients; RF–/RA, RF-seronegative RA patients; OA, osteoarthritis; PEG, polyethylene glycol; CHX, cycloheximide. Back

Received for publication July 7, 2006. Accepted for publication March 27, 2007.


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 Discussion
 Disclosures
 References
 

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