Increased expression and release of functional tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by T cells from lupus patients with active disease

Clinical Immunology 117 (2005) 48 – 56 www.elsevier.com/locate/yclim

Increased expression and release of functional tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by T cells from lupus patients with active disease Violeta Rusa,*, Valentina Zernetkinaa, Roman Puliaeva, Cornelia Cudricib, Susan Mathaia, Charles S. Viaa a

Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Maryland at Baltimore, MSTF Building, Room 8-34, 10 S Pine Street, Baltimore, MD 21201, USA b Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201, USA Received 24 February 2005; accepted with revision 4 May 2005 Available online 15 June 2005

Abstract Increased expression of TRAIL in membrane-bound and soluble form in patients with systemic lupus erythematosus (SLE) has been previously reported. In this study, we characterized the upregulation of T-cell-associated and soluble TRAIL (sTRAIL) in vivo and the modulation of TRAIL expression and soluble protein release in vitro following T cell activation and IFNa exposure. The expression of membrane-bound TRAIL as determined by flow cytometry was higher on CD4+ and CD8+ T cells from lupus patients compared to controls, particularly on activated CD69+CD8+ T cells. Similarly, sTRAIL levels determined by ELISA were significantly elevated in serum from patients with active SLE and correlated with levels of IFNa. In vitro, both T-cell-associated and sTRAIL were maximally induced by T cell activation plus IFNa in patients and controls. By Western blot analysis, sTRAIL was detected in sera in both the monomeric and multimeric, functional form. Both forms of TRAIL were functional in vitro as determined by Annexin V staining and 51Cr release assay but the apoptotic activity of membrane TRAIL was 2.5-fold higher compared to that of sTRAIL. These results indicate that IFNa-induced enhancement of TRAIL expression and of TRAIL-mediated apoptosis may amplify the abnormal apoptotic process in SLE. D 2005 Elsevier Inc. All rights reserved. Keywords: Tumor necrosis factor-related apoptosis-inducing ligand; IFNa; Apoptosis; Systemic lupus erythematosus; Disease activity

Introduction Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family with pro-apoptotic activity [1]. In the immune system, TRAIL is expressed on the surface of activated T lymphocytes [2], IFNa-stimulated monocytes [3– 6], dendritic cells [7] and IFNg-stimulated NK cells [8]. TRAIL can interact with five different receptors. TRAIL-R1/(DR4) and TRAIL-R2/(DR5) are capable of transmitting a death signal [9], while the other three receptors TRAIL-R3

* Corresponding author. Fax: +1 410 706 0231. E-mail address: [email protected] (V. Rus). 1521-6616/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2005.05.001

(DcR1), TRAIL-R4 (DcR2), and the soluble receptor, osteoprotegrin, act as decoy receptors blocking TRAILinduced apoptosis. Among cytokines, IFNs, especially type I IFNs, have the ability to enhance expression of surface TRAIL on immune cells. Similar to other members of the TNF family such as CD40L, FasL, and TNF, TRAIL can be released in soluble form and low amounts can be measured in sera of normal controls. TRAIL binding to its death signaling receptors mediates apoptosis of malignant transformed cells but also of HIV-infected lymphocytes, normal monocytes, neutrophils [10], and macrophages [11 – 14]. In addition to its apoptotic activity, TRAIL plays an important role in immune homeostasis through non-apoptotic pathways. In vitro studies have demonstrated the ability of TRAIL to

V. Rus et al. / Clinical Immunology 117 (2005) 48 – 56

inhibit T cell activation, cell cycle progression, and IFNg and IL-4 production [15]. Furthermore, in vivo treatment of gld/gld mice [16] with recombinant adenoviral vector encoding TRAIL inhibited the production of autoantibody. Conversely, TRAIL blockade by soluble TRAIL-R2 or anti-TRAIL mAb exacerbated murine experimental autoimmune encephalomyelitis [17], collagen-induced arthritis [18] and enhanced the autoantibody responses in gld/gld mice [16,19] without affecting the rate of apoptosis, suggesting that TRAIL – TRAIL R binding may be an important downregulatory mechanism in T-cell-driven responses. Recent studies in SLE patients have demonstrated increased TRAIL mRNA in peripheral blood mononuclear cells (PBMC) and upregulated surface expression on T cells [10,20 –22]. Furthermore, T-cell-associated TRAIL induced apoptosis of monocytes and neutrophils from SLE patients [10,22], suggesting that TRAIL may be important in modulating disease expression in SLE patients. To clarify the role of TRAIL in SLE patients, we examined both T-cell-associated and sTRAIL in vivo and in vitro using cells and sera from SLE patients with a wide range of clinical manifestation. Our results demonstrate that increased serum levels of sTRAIL are positively correlated with disease activity and with IFNa levels. Moreover, our in vitro data support the idea that characteristic features of SLE, such as T cell activation and IFNa, promote TRAIL-mediated apoptosis in vivo.

Methods Subjects Thirty-four patients fulfilling ACR criteria for SLE [23] followed at the Lupus Program of the University of Maryland School of Medicine were recruited for this study. Disease controls consisted of connective tissue disease patients with rheumatoid arthritis (n = 7), psoriatic arthritis (n = 2), Sjo¨ gren syndrome (n = 2), and polymyositis (n = 1). Healthy sex- and age-matched controls (n = 26) were also included. SLE activity was assessed by the SELENA – SLEDAI [24] and patients with SELENA – SLEDAI4 were considered to have active disease. The characteristics of the patients and controls are summarized in Table 1.

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Table 1 Demographic characteristics, disease activity index, and medications in the studied subjects Characteristic

SLE patients (n = 34)

Healthy controls (n = 26)

Disease controls (n = 12)

Female/Male Age mean (range), years

34/0 38 (19 – 66)

26/0 41 (25 – 53)

10/2 49 (19 – 73)

Race Caucasian African American Asian

8 25 1

7 19 0

SLEDAI score 4 – 40 0–3

14 20

N/A N/A

Treatment None Prednisone HCQ Immunosuppressors

0 21 17 5

26 0 0 0

8 4 0

N/A N/A

0 5 4 6

HCQ = hydroxychloroquine; N/A = not applicable.

R1: Fc and TRAIL R2: Fc chimera, was obtained from Alexis Biochemical (San Diego, CA). PBMC and T cell isolation and culture Peripheral blood mononuclear cells were isolated from heparinized venous blood by Ficoll density-gradient centrifugation. T cells were separated by the Rosette purification method (Stem Cell Technologies, Vancouver, British Columbia, Canada) followed by Ficoll-Paque separation gradient as recommended by the manufacturer. The purity of isolated human T cells was >95% as tested by flow cytometry with anti-CD3 monoclonal antibodies. Immediately after purification, 4  106 T cells were cultured in Iscove’s modified Dulbecco’s medium (Gibco, Grand Islands, NY, USA) supplemented with l-glutamine, sodium pyruvate, non-essential amino acids, penicillin/streptomycin, 2ME, and 5% human serum. Cells were either untreated or stimulated with 10 Ag/ml anti-CD3, 1 Ag/ml CD28 and, where indicated, 200 U/ml IFNa. At 48 h, SN were harvested and frozen until testing. T cells were further processed for immunofluorescent staining or cytotoxicity assays. Determination of serum levels of sTRAIL

Antibodies and reagents The following mouse anti-human antibodies were obtained from BD Pharmingen (San Diego, CA): FITCanti-CD3, FITC-anti-CD4, FITC-anti-CD8, FITC-antiCD69, PE-anti-human TRAIL, PE mouse IgG1, purified anti-CD3, anti-CD28. Purified recombinant human IFNa was obtained from PeproTech Inc. (Rocky Hill, NJ). Recombinant human Killer TRAIL (rKiller TRAIL), TRAIL

The concentration of sTRAIL in cell-free supernatants and sera obtained from patients and controls was measured using a commercial ELISA kit (Diaclone Research, Besancon, France) according to the manufacturer’s instruction. The lower limit of detection of the assay is 64 pg/ml. To eliminate day-to-day interassay variability, all serum samples were stored at 70-C and processed on the same day. Serum IFNa was determined

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using multispecies human IFNa ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ). Immunofluorescent staining Fresh PBMC or in vitro stimulated and unstimulated T cells were washed with standard buffer (PBS, 2% BSA, 0.1% sodium azide) incubated for 30 min at 4-C with 0.1 –1 Ag/ml fluorochrome-conjugated mAb following the manufacturer’s recommendations, washed three times with standard buffer, then fixed in 1% paraformaldehyde and analyzed in a FACScan flow cytometer (BD Biosciences, Mountain View, CA). TRAIL expression was determined on gated CD3+ cells and CD4+CD69+ and CD8+CD69+ cells. Data collected from 10,000 cells are reported as either percentage of positive cells or mean channel fluorescence intensity (MCF). Immunoprecipitation and Western blot analysis of sTRAIL from serum Human serum (500 Al) from SLE patients or healthy donors were centrifuged at 10,000g. Aliquots were then immunoprecipitated at 4-C overnight with 2 Ag of antiTRAIL antibody (Santa Cruz Biotechnology, Santa Cruz, CA) that had been incubated with Protein A/G PLUSAgarose beads. After removal of supernatant, the samples were analyzed by Western blot under reducing and nonreducing conditions. Samples were washed with PBSTween 20 (0.05%), boiled for 5 min in reducing buffer (5% sodium dodecylsulfate [SDS]-mercaptoethanol) or for 3 min in nonreducing buffer (NuPage lithium dodecylsulfate; Invitrogen, Carlsbad, CA), and then subjected to SDSPAGE. Recombinant human sTRAIL (rhsTRAIL) from Biomol (Plymouth Meeting, PA) and R&D Systems (Minneapolis, MN) was used as positive control for reducing and, respectively, nonreducing Western blot conditions. Proteins were then electroblotted onto nitrocellulose membranes Immobilon-P (Millipore, Billerica, Ma) then blocked for 1 h with 1% BSA in 0.1% Tween20 in Tris-buffered saline. The blots were then washed 2 times with 0.05% Tween-20 in TBS and incubated overnight with monoclonal mouse anti-human TRAIL antibody (BD Pharmingen) at a dilution of 1:300. After 3 washings, the blots were incubated with horseradish peroxidaseconjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology) and developed using enhanced chemiluminescence (Pierce Chemical Co, Rockford, IL). The blots were then exposed to Blue Bio Film (Denville Scientific Inc., Metuchen, NJ). Supernatant (SN) induction of apoptosis T cells from six patients and six controls were cultured with plate-bound anti-CD3 mAb (10 Ag/ml) and anti-CD28 mAb (1 Ag/ml) in the presence or absence of IFNa (200 U/ ml) and supernatants harvested at 48 h. SN were then

concentrated by Amicon Ultra-4 Centrifugal Filter Devices (Amicon Inc., Beverly, MA, USA). TRAIL-sensitive Jurkat J32 cells were seeded at a density of 1  106 and incubated for 48 h with media conditioned with IFNa alone, 5 ng/ml rKiller TRAIL, or concentrated supernatants at final sTRAIL concentration of 5 ng/ml (as measured by ELISA). Where indicated, blocking was performed using chimeric TRAIL-R1:Fc at 5 Ag/ml and TRAIL-R2:Fc at 0.5 Ag/ml. Cells undergoing apoptosis were identified by Annexin V staining (BD Pharmingen, San Diego, CA). Cytotoxicity assays For cell-mediated cytotoxicity assays, purified T cells from three SLE patients were used as effector cells and were incubated with 51Cr-labeled Jurkat cells (104) at the indicated E/T ratio in U-bottomed 96-well microtiter plates. For direct cytotoxicity assays, 51Cr-labeled Jurkat cells (104) were cultured in the presence of concentrated supernatants of stimulated T cells at a final concentration of sTRAIL of 5 ng/ml. Cr release was determined 18 h later as previously described [22]. TRAIL-R1:Fc at 5 Ag/ml and TRAIL-R2:Fc at 0.5 Ag/ml were used to block TRAIL-induced apoptosis of Jurkat cell lines. Statistical analysis Two-tailed unpaired Student’s t test and Pearson correlation coefficient test were used for statistical evaluation. A P value below 0.05 was considered statistically significant. Most results are expressed as mean T standard deviation (SD).

Results Increased expression of TRAIL on activated CD4+ and CD8+ T cells from lupus patients Previous studies from our group and from others have reported increased TRAIL mRNA expression in PBMC and increased TRAIL protein expression on the surface of T cells in lupus patients [20 –22]. Because T lymphocytes upregulate TRAIL expression upon TCR engagement, we investigated whether TRAIL expression is upregulated on in vivo activated T cells from lupus patients. We first determined the expression of surface TRAIL on freshly isolated T cell subpopulations from 10 patients and 10 healthy controls. As shown in Fig. 1, a higher percentage of CD4+ and CD8+ T cells from lupus patients expressed TRAIL compared to controls (Fig. 1). The difference was greater on CD8+ (15.9% T 5.3 in patients versus 5.2% T 1.5 in controls, P = 0.04) than on CD4+ T cells (9.3% T 2.1 versus 4.7% T 1.2, P = 0.06). In addition, MCF for TRAIL was higher on CD4+ and CD8+ T cells from patients compared to controls and more so on CD4+ (114.8 T 18.1 in

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Fig. 1. TRAIL expression is increased on both CD4+ and CD8+ T cells from SLE patients. PBMC from SLE patients and controls were assessed by flow cytometry as described in Methods. Results are shown as percent positive TRAIL cells (group mean T SD) for (A) total CD4+ and CD8+ T cells or for (B) activated CD69+CD4+ and CD69+CD8+ T cells (n = 10 SLE, 10 controls). Individual TRAIL expression is shown for (C) CD4+ or (D) CD8+ T cells from a single patient with high cell surface expression and from a normal control.

patients versus 62.9 T 6.2 in controls, P = 0.01) than on CD8+ T cells (32.4 T 6.0 versus 27.3 T 1.4, P = 0.06). When activated CD69+CD4+ and CD69+CD8+ T cells were analyzed, the percentage of TRAIL+ cells was significantly higher on CD69+CD8+ T cells from SLE patients compared to controls (24% T 0.9 vs. 1.9% T 1.1, P = 0.03). A similar trend was noted for CD69+CD4+ cells (60% T 20 vs. 39% T 8, P = 0.1); however, the difference was not statistically significant. These results are in agreement with those of Kaplan et al. who studied resting and CD25+ and CD29+ activated T cells from SLE patients [22].

controls exhibited sTRAIL levels similar to SLE patients (mean 2219 T 738 pg/ml; P = 0.02 vs. controls). We then examined whether any of the variability of sTRAIL levels could be explained by SLE disease activity. As seen in Fig. 3, patients with active disease (SELENA – SLEDAI  4) had significantly higher mean values of sTRAIL (2475 T 836 pg/ml) when compared to patients with inactive disease (1786 T 850 pg/ml, P = 0.02) or healthy controls (1595 T 697 pg/ml, P = 0.002). Moreover, serum sTRAIL levels also correlated with SELENA –

Elevated levels of sTRAIL in sera from patients with SLE Similar to other members of the TNF family, TRAIL exists in both membrane-bound and soluble form [25,26]. To determine whether the increase in membrane-bound TRAIL expression in SLE patients seen in Fig. 1 is accompanied by an increase in the released protein, sTRAIL levels were measured by ELISA in serum specimens from lupus patients, disease controls, and healthy volunteers. As seen in Fig. 2, despite variability in sTRAIL serum levels, high levels (>2000 pg/ml) were seen in a greater proportion of SLE patients (16/34) compared to controls (5/26). Moreover, the mean sTRAIL concentration in SLE sera was 2107 T 893 pg/ml compared to 1595 T 697 pg/ml in healthy controls (P = 0.01). Elevated serum sTRAIL levels were not specific to SLE as connective tissue disease

Fig. 2. Elevated levels of sTRAIL in SLE sera. Serum samples from 26 healthy controls, 12 disease control patients with a variety of connective tissue diseases, and 34 SLE patients were tested to determine the concentration of sTRAIL by ELISA. Levels of sTRAIL were significantly higher ( P = 0.01) in serum samples from SLE patients compared to healthy controls but not to disease controls.

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released in multimeric form, thus likely to be functionally active. Membrane and sTRAIL are upregulated by TCR and IFNa stimulation

Fig. 3. Relationship between sTRAIL serum concentration and SLE disease activity. (A) Thirty-four SLE patients were classified as having either active (n = 14; SELENA – SLEDAI  4) or inactive disease (n = 20; SELENA – SLEDAI < 4) as defined in Methods. Sera from these patients and 26 healthy controls were then tested to determine the concentration of sTRAIL by ELISA. (B) Correlation between concentration of serum sTRAIL (as indicated on the ordinate in pg/ml) and SELENA – SLEDAI (as indicated on the abscissa) in each of the 26 SLE patient samples. The line represents the best fit correlation between the two variables (Pearson r = 0.5, P = 0.001).

SLEDAI as a continuous measure (Pearson r = 0.5, P = 0.001). No significant difference was noted between patients with inactive disease and healthy controls. In addition, no correlation was found between specific manifestations of disease activity and sTRAIL levels. Thus, increased TRAIL expression, as either membrane or soluble form, is a feature of SLE patients with active disease.

In addition to T cell activation, type I IFNs act as specific stimuli for the upregulation of membrane-expressed TRAIL and/or sTRAIL release from a variety of immune cells. On T cells, IFNa has the ability to provide a costimulatory signal to anti-CD3-induced surface TRAIL upregulation [4]. To determine whether T cells from lupus patients upregulate TRAIL in response to combined TCR and IFNa stimulation in vitro, surface expression of TRAIL was determined on purified anti-CD3/CD28-stimulated CD3+ cells, with or without added IFNa. Consistent with previous observations [3,4], a slight but statistically insignificant increase in both membrane and sTRAIL was seen with IFNa alone. Patients and controls both demonstrated TRAIL upregulation on stimulated T cells compared to media alone but the change was statistically significant only in controls, likely as a result of the large variability and lower relative increase in TRAIL expression among SLE patients (Fig. 5). Several studies have suggested that surface-bound and sTRAIL may be differentially regulated depending on the stimuli used in vitro [3,27]. To assess whether stimulation through TCR and IFNa increases sTRAIL release along with surface TRAIL upregulation on T cells, we determined sTRAIL levels in supernatants of T cells stimulated as above. A significant increase of sTRAIL was seen in supernatants of T cells from both patients and controls when stimulated with combined anti-CD3/CD28 and IFNa compared to media alone (SLE: 688 T 90 pg/ml vs. 510 T 44 pg/ml, P = 0.007; controls: 910 T 123 pg/ml vs. 525.5 T 45 pg/ml, P = 0.03). As seen with membrane TRAIL, sTRAIL values following T cell stimulation alone were not statistically significant in SLE patients. Only with the addition of IFNa to TCR stimulation did sTRAIL values

Molecular size characteristics of sTRAIL in sera To confirm the presence of the soluble form of TRAIL in sera, we initially performed immunoprecipitation of serum samples from 3 normal subjects and 3 SLE subjects followed by Western blot under reducing conditions. As reported in Fig. 4A, sTRAIL was detectable in both SLE and healthy control sera as a 24-kDa band, similar in migration pattern with the monomeric rhsTRAIL used as positive control. In order to investigate the aggregation state of sTRAIL in serum, Western blot analyses were also performed under nonreducing conditions. As shown in Fig. 4B, two bands of approximately 48 and 66 kDa similar to the soluble trimeric rhsTRAIL control were detectable in serum of patients and controls, suggesting the presence of the multimeric form of the protein. Altogether, these results suggest that sTRAIL in SLE and normal control sera are

Fig. 4. Immunoblot analyses of sTRAIL immunoprecipitated from control and SLE sera. Sera from controls (lanes 1 – 3) and patients (lanes 4 – 6) were immunoprecipitated with anti-TRAIL mAb and analyzed for the presence of sTRAIL by Western blot under reducing (A) and nonreducing conditions (B) as described in Methods. Lane 7 represents recombinant human sTRAIL that served as positive control.

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Fig. 5. Enhancement of TRAIL cell surface expression and protein release from purified T cells of SLE patients and controls stimulated with CD3/ CD28 and/or IFNa. (A) Flow cytometry analysis of surface expression of TRAIL on purified T cells from six healthy donors and six lupus patients cultured for 48 h in the presence of 10 Ag anti-CD3 and 1 Ag anti-CD28 with or without 200 IU/ml IFN-a. (B) Supernatants harvested from purified T cells cultured as above were tested for sTRAIL release by ELISA. The mean values T SD of sTRAIL release from 6 patients and 6 controls are shown. *P < 0.05 compared to media.

increase significantly. The addition of IFNa resulted in no further enhancement of sTRAIL levels in controls, implying that the response to TCR stimulation alone is probably maximal.

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CD28+ IFNa-stimulated T cells were used at a final sTRAIL concentration of 5 ng/ml (as determined by ELISA). rKiller TRAIL, a recombinant soluble form of TRAIL with biological activity resembling the membranebound form served as the positive control and was also used at 5 ng/ml. As shown in Fig. 7A, rKiller TRAIL induced an approximate fourfold increase in the percentage of apoptotic cells over untreated cells which was inhibited by approximately 52% by the TRAIL-specific inhibitors, TRAIL-R1:Fc and TRAIL-R2:Fc. Much lower apoptosis was seen with sTRAIL containing supernatants from both controls and SLE patients (controls: 1.6-fold increase; SLE patients 1.7-fold increase) and was TRAIL specific as shown by combined TRAIL-R1: Fc/TRAILR2: Fc blocking. No apoptosis of Jurkat cells was induced by IFNa alone. The low levels of apoptosis induced by sTRAIL raise the possibility that membrane TRAIL may be more effective. To address this question, we compared the ability of T cell membrane TRAIL and sTRAIL to induce lyses of 51Cr-labeled Jurkat cells. sTRAIL was used at concentrations which approximate the highest levels detected in serum from lupus patients. T cells from three SLE patients exhibited a mean lysis of Jurkat cells of 50% which was primarily TRAIL mediated (74% inhibitable with TRAIL-R1:Fc/TRAIL-R2:Fc). sTRAIL from supernatants of stimulated lupus T cells induced much lower levels (20%) of Jurkat cell lyses which were partially inhibited (55%) by TRAIL blockade (Fig. 7B). These data demonstrate that although both membranebound and sTRAIL exhibit biologic activity in vitro, membrane TRAIL is more effective in inducing apoptosis in susceptible targets than is sTRAIL.

Discussion Serum sTRAIL levels correlate with levels of IFNa in patients with lupus

In this study, we demonstrate increased soluble and Tcell-associated TRAIL in lupus patients with active

To address the relationship between sTRAIL and IFNa in SLE, we quantitated by ELISA serum levels of multispecies IFNa in 21 lupus patients and 12 controls. IFNa was detectable above background in 16/21 serum specimens from SLE patients and in none of the controls (Fig. 6). A significant correlation was observed between serum IFNa and sTRAIL levels (Pearson r = 0.45, P = 0.002) in SLE patients. Both membrane TRAIL and sTRAIL induce apoptosis in vitro To ascertain whether the levels of sTRAIL detected in SLE sera are biologically active, we tested the ability of T-cell-derived supernatants from six lupus patients and six controls to induce apoptosis of a TRAIL susceptible target (Jurkat cells). Supernatants obtained from anti-CD3/

Fig. 6. Correlation of serum sTRAIL with levels of IFNa. Shown are the relationships between concentrations of sTRAIL and IFNa determined by ELISA in serum samples from 21 SLE patients. The line represents the best fit correlation between the two variables (Pearson r = 0.45, P = 0.002).

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Fig. 7. Membrane-bound and sTRAIL display apoptotic activity in vitro. (A) Apoptosis rate of Jurkat cells was assessed by Annexin-V/PI staining after a 2-day culture in the presence of media alone, 5 ng/ml rKiller TRAIL, and T cell-derived anti-CD3/CD28+ IFNa-stimulated supernatant from six patients and six controls. Cell incubation was carried out in the absence or presence of a combination of 5 Ag/ml TRAIL-R1: Fc and 0.5 Ag/ml TRAIL-R2: Fc (R1/R2: Fc). Cell-free culture medium containing IFNa was used as a control. Results represent mean values T SD for the patient and control group and of triplicates for media and IFNa controls. (B) Cytotoxic activity of T-cell-associated membrane TRAIL and sTRAIL against Jurkat cell line. T cells from three SLE patients were tested for lyses of 51Cr-labeled Jurkat cells by an 18 h 51Cr-release assay at an E/ T ratio of 10 in the absence or presence of a combination of 5 Ag/ml TRAIL-R1:Fc and 0.5 Ag/ml TRAIL-R2:Fc (R1/R2:Fc). Supernatants of anti-CD3/CD28+ IFNa-stimulated T cells from same patients were used in the 51Cr-release assay at a final concentration of sTRAIL of 5 ng/ml. Results represent mean values T SD.

disease compared to healthy controls. Furthermore, sTRAIL levels correlated significantly with disease activity as measured by SELENA –SLEDAI. Similar to other reports, we found no association between increased levels of sTRAIL and any specific feature of disease activity [28]. Although Matsuyama et al. [10] have reported increased levels of sTRAIL in lupus patients with neutropenia compared to patients with normal neutrophil counts, only a few of our patients with active disease had neutropenia, supporting the idea that increased sTRAIL is associated with multiple manifestations of disease activity and not with a specific phenotype. Furthermore, similar to previous reports of membranebound TRAIL [22,29], our finding of increased sTRAIL was not specific for lupus and was present in other conditions characterized by immune system activation.

The correlation between serum levels of sTRAIL and IFNa in lupus patients was not unexpected. Our group [20] and others [21] have identified TRAIL as one of the upregulated genes belonging to the IFNa signature in PBMC from SLE patients. The capacity of type I IFNs to induce membrane expression and/or sTRAIL release in activated T cells, monocytes, neutrophils, and NK cells has been previously established [3,4,10,27]. Furthermore, on normal T cells, the role of IFNa and h in modulating TRAIL expression is unique, as other cytokines such as IFNg and IL-15 can upregulate mRNA [10,30] but not surface expression of TRAIL [4]. Our data extend to SLE previous in vitro observations suggesting a costimulatory role of IFNa in the upregulation of surface TRAIL on normal activated T cells [4]. We also demonstrate a similar role for IFNa in sTRAIL release. Our observation that in vitro lupus patients exhibit impaired upregulation of T cell surface TRAIL and sTRAIL protein release compared to normals, yet paradoxically exhibit higher levels of both in vivo, is reminiscent of the wellrecognized paradoxical finding that SLE T cells exhibit impaired function in vitro but are hyperactive in vivo [31,32]. In addition, other cells such as monocytes, NK cells, and neutrophils may release sTRAIL in response to IFNa and/or cell activation in vivo [3,27], thereby contributing to the increased serum levels. A link between TRAIL and IFNa has been previously identified. For example, increased circulating levels of sTRAIL have been reported after treatment with IFNa in patients with chronic myeloid leukemia [27] or following treatment with IFNh in patients with multiple sclerosis [33]. Interestingly, serum IFNa levels found in the majority of SLE patients in our group were equal to or greater than those reported in IFNa-treated patients. Of interest is the observation that increased levels of sTRAIL correlate with the responsiveness to IFNh treatment in multiple sclerosis [33], leading to the proposal that TRAIL could be used as a prognostic therapeutic marker in this autoimmune disease. As IFNa has emerged as a new target for therapy in SLE (reviewed in [34]) and biologics that block IFNa are in development, it will be important to determine whether increased sTRAIL levels in SLE patients will identify potential candidates for IFNa blockade and whether a decrease in sTRAIL correlates with treatment response. Our results also suggest a mechanism by which IFNa may exacerbate lupus. It has been previously shown that IFNa is pro-apoptotic. IFNa promotes FasL-mediated apoptosis by NK cells [35] and TRAIL-mediated cytotoxicity of tumor cells by T cells, monocytes, or NK cells [4,36,37]. Similarly, in our study, T-cell-associated TRAIL from lupus patients was a potent inducer of apoptosis of TRAIL-sensitive Jurkat cells. Although normal cells are generally resistant to TRAIL-mediated apoptosis, recent studies have demonstrated that neutrophils and monocytes from lupus patients are sensitive to TRAIL [10,22]. Thus,

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by enhancing TRAIL expression and TRAIL-mediated apoptosis of susceptible cells, such as neutrophils and monocytes, IFNa may simultaneously increase the apoptotic load and decrease the number of cells involved in apoptotic clearance, both of which are characteristic features of SLE [22,38]. The relative contribution in vivo of sTRAIL compared to membrane-associated TRAIL is not completely clear at present. Previous work has shown that sTRAIL at low concentrations comparable to those found in serum of healthy volunteers and lupus patients with inactive disease had no effect in vitro on T cell apoptosis or proliferation [15,39]. In contrast, several of SLE sera in our study contained >4000 pg/ml, a level compatible with concentrations that we observed to be functional in vitro. Furthermore, the detection of multimeric forms of sTRAIL in serum suggests that secreted TRAIL can mediate apoptotic effects in SLE. However, the finding in our study that the apoptotic ability of sTRAIL is lower than that of the cell-bound form suggests that, similar to FasL and TNFa, the proteolytic cleavage of TRAIL might be a mechanism by which the more active membrane-bound form of the molecule is downregulated [26,40,41]. Lastly, in addition to its pro-apoptotic effects, nonapoptotic activities of lymphocyte-associated TRAIL have been described, and although conflicting, they suggest a complex role for TRAIL in SLE. For example, reverse signaling through TRAIL was reported to result in enhanced proliferation of T cells from both normals and SLE patients [42], whereas TRAIL binding and signaling through its receptors has been reported to decrease T cell proliferation, and IFNg and IL-4 production by autoantigen-specific T cell lines [15]. In gld/gld lupus prone mice, TRAIL was shown to suppress autoantibody production [16] and lymphocyte proliferation [19]. An agonistic antibody mimicking the activity of native TRAIL is currently being tested in preliminary clinical trials in patients with malignancies; thus, it will be important to fully clarify the biologic role of TRAIL in SLE in order to adequately assess the therapeutic potential of TRAIL biologics in SLE. In summary, we have demonstrated increased T cell surface expression and soluble protein release of TRAIL in SLE patients with active disease, most likely reflecting in vivo TCR engagement and IFNa stimulation. Enhanced TRAIL-mediated apoptosis by surface-bound and sTRAIL may be an important intermediary in the link between IFNa and systemic autoimmunity. These results lend support to further studies evaluating the potential use of TRAIL as a biomarker and therapeutic target in patients with SLE.

Acknowledgments Supported by grants from the NIH (K23 AR02135-01) and by an Arthritis Foundation Investigator Award.

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