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Sphingosine Kinase 1–Mediated Inhibition of Fas Death Signaling in Rheumatoid Arthritis B Lymphoblastoid Cells

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Sphingosine Kinase 1–Mediated Inhibition of Fas Death Signaling in Rheumatoid Arthritis B Lymphoblastoid Cells ARTHRITIS & RHEUMATISM Vol. 54, No. 3, March 2006, pp 754–764 DOI 10.1002/art.21635 © 2006, American College of Rheumatology Sphingosine Kinase 1–Mediated Inhibition of Fas Death Signaling in Rheumatoid Arthritis B Lymphoblastoid Cells Xiujun Pi, Shi-Yu Tan, Michael Hayes, Liqun Xiao, James A. Shayman, Song Ling, and Joseph Holoshitz Objective. It is becoming increasingly apparent increased levels of S1P. Real-time PCR analysis showed that B cells play an important role in the pathogenesis higher SPHK-1 mRNA expression levels in RA patients of rheumatoid arthritis (RA). Due to the scarcity of B compared with paired controls. Increased SPHK-1 (but cells in RA, it has been technically difficult to function- not SPHK-2) mRNA levels were observed in synovial ally characterize B cell apoptosis in this disease. As a tissue from RA patients. Competitive inhibitors of necessary first step to identify candidate aberrations, we SPHK reversed the resistance of RA LCLs to Fas- investigated Fas-mediated signaling events in immortal- induced apoptosis. Additionally, resistance to Fas- ized peripheral blood B lymphoblastoid cell lines mediated signaling was reversed by siRNA oligonucleo- (LCLs) from patients with RA and controls. tides specific for SPHK-1 but not by oligonucleotides Methods. Cell death was determined by the MTS specific for SPHK-2. assay, and apoptosis was detected by the TUNEL assay Conclusion. These findings demonstrate disease- and DNA laddering. Proteolytic activation of caspase 3 specific resistance to Fas-mediated death signaling in was determined by immunoblotting, and its enzymatic patients with RA and implicate increased SPHK-1 ac- activity was determined by a fluorometric technique. tivity as the cause of this aberration. Messenger RNA (mRNA) expression was quantified by real-time polymerase chain reaction (PCR) analysis. Rheumatoid arthritis (RA) is a systemic disease The functional role of sphingosine kinase (SPHK) was characterized by infiltration of inflammatory cells into determined by measuring its enzymatic activity, by the synovium and hyperplasia of the synovial lining cells quantifying the levels of its product, sphingosine that lead to destruction of adjacent cartilage and bone 1-phosphate (S1P), and by investigating the ability of (for review, see ref. 1). It has been previously postulated N,N- the SPHK inhibitor dimethylsphingosine and that either aberrant apoptosis or migration of cells from isozyme-specific small interfering RNA (siRNA) oligo- the peripheral blood into the synovial compartment may nucleotides to reverse signaling aberrations. be responsible for pathologic stockpiling of cells in the Results. LCLs from patients with RA displayed rheumatoid pannus (2–7). disease-specific Fas-mediated signal transduction im- Over the past several years, interest in the patho- pairment with consequent resistance to cell death. RA genic role of B lymphocytes in RA has reemerged (for LCLs displayed high constitutive SPHK activity and review, see ref. 8). In addition to the realization that Supported by grants R01-AI-47331, R01-AR-46468, P60-AR- antibodies do play important roles in experimental mod- 20557, and P30-AR-48310 from the NIH, and by a Biomedical Science els of RA and in the human disease, there is increasing Grant from the Arthritis Foundation. Dr. Pi’s work was supported by evidence that B lymphocytes accumulate and mature in Postdoctoral Training Grant T32-AR-07080 from the NIH. Xiujun Pi, MD, PhD, Shi-Yu Tan, MD, Michael Hayes, BSc, the inflamed synovium, where they can form ectopic Liqun Xiao, MD, James A. Shayman, MD, Song Ling, PhD, Joseph germinal centers (9–11) and activate T cells (12). Addi- Holoshitz, MD: University of Michigan Medical Center, Ann Arbor. tionally, experimental treatments with anti-CD20 anti- Drs. Pi and Tan contributed equally to this work. Address correspondence and reprint requests to Joseph Ho- bodies have shown promise (13) and further support the loshitz, MD, 5520D MSRB1, Box 0680, University of Michigan, 1150 growing consensus that B lymphocytes play an important West Medical Center Drive, Ann Arbor, MI 48109-0680. E-mail: role in the pathogenesis of RA. The propensity of B [email protected]. Submitted for publication August 25, 2005; accepted in lymphocytes to accumulate in the synovium has been revised form November 10, 2005. attributed to the local trophic influence of resident 754 DISEASE-ASSOCIATED DYSREGULATION OF SPHK 755 Table 1. Clinical features of the patients with rheumatoid arthritis* Patient/sex/ age, years Disease duration RF Nodules Erosions Therapy HLA–DRB1 1/M/62 23 years ϩϩ ϩGold, MTX 0401/0404 2/M/46 8 years ϩϩ ϩAZA, gold, HCQ, MTX, SSZ 0401/0404 3/F/48 6 years ϩϩ ϩHCQ, MTX 0401/0404 4/F/42 6 months – – – NSAID 0401/11 5/M/44 11 years ϩϩ – MTX, prednisone 0401/0404 6/F/62 33 years ϩϩ ϩAZA, gold, HCQ, MTX, SSZ 0401/0401 7/M/54 10 years ϩϩ ϩGold 0401/15 8/F/45 7 years ϩϩ ϩMTX, NSAID 0401/17 9/M/64 16 years – – ϩ NSAID 0101/0701 10/F/53 5 months ϩ – – NSAID 17/17 11/F/21 14 months ϩ – – Gold, HCQ, NSAID 0101/0404 12/M/72 19 years – – ϩ NSAID, prednisone 0401/14 13/M/52 4 months ϩ – ϩ NSAID, prednisone 0101/15 14/F/34 11 years ϩ – ϩ NA 0401/1201 15/F/53 9 years ϩ – ϩ NA 0401/1104 16/M/44 NA ϩϩ ϩNA 0101/0301 17/F/NA NA NA NA NA NA 0101/1302 18/F/37 5 years ϩϩ ϩNA 0401/0401 19/F/36 6 years ϩϩ ϩNA 0404/1401 20/M/32 10 years – – ϩ NA 0401/0701 21/F/62 13 years ϩϩ ϩNA 0401/0701 22/F/65 3 years ϩ – – NA 0401/0401 23/M/58 6 months ϩ – – NSAID 0405/11 *RFϭ rheumatoid factor; MTX ϭ methotrexate; AZA ϭ azathioprine; HCQ ϭ hydroxychloroquine; SSZ ϭ sulfasalazine; NSAID ϭ nonsteroidal antiinflammatory drug; NA ϭ not available. synoviocytes (9,10). However, what the mechanisms thematosus [SLE], 10 with juvenile RA [JRA], 3 with auto- governing their homing to the synovium are, and immune thyroiditis, and 5 with insulin-dependent diabetes whether B cells are intrinsically resistant to programmed mellitus [IDDM], as well as 29 healthy controls) was used in this study. The RA group (58% women and 42% men, mean Ϯ cell death, are questions that are presently unanswered. SD age 49.2 Ϯ 11.9 years) and the control group (61% women There is ample evidence that B cell Fas-mediated and 39% men, mean Ϯ SD age 45 Ϯ 12.3 years) did not differ cell death plays a key role in the maintenance of self demographically. The salient clinical features of the RA group tolerance, and that failure of this mechanism can lead to are shown in Table 1. A panel of LCLs from 10 RA-discordant autoimmunity (for review, see ref. 14). However, due to monozygotic twin pairs (7 female pairs and 3 male pairs, the marked scarcity of peripheral B lymphocytes in RA mean Ϯ SD age 45.2 Ϯ 10.7 years) was used in some (15,16), little is known about Fas-mediated cell death of experiments. Synovial tissues obtained during joint replace- ment surgery in 14 patients with RA and 9 patients with B lymphocytes in this disease. osteoarthritis (OA) were used to quantify synovial tissue In this study, we investigated the efficiency of SPHK messenger RNA (mRNA) expression. Fas-mediated death signaling in peripheral blood B Cells and culture conditions. LCLs were prepared lymphocytes. In an attempt to overcome the scarcity of from peripheral blood B cells, using a standard technique (19), peripheral B lymphocytes in RA, we used immortalized and were cultured in supplemented RPMI 1640 containing lymphoblastoid cell lines (LCLs). Our data indicate that 10% heat-inactivated fetal bovine serum (Irvine Scientific, Santa Ana, CA). In all experiments, test and control cells LCLs from patients with RA are uniquely resistant to were obtained from lines that had been maintained in iden- Fas-mediated cell death. The aberration is attributable tical tissue culture conditions, with identical cell density and to overactivity of sphingosine kinase 1 (SPHK-1), with viability. resultant overproduction of sphingosine 1-phosphate Induction of apoptosis. Fas-mediated apoptosis was ϫ 4 (S1P), a sphingolipid previously shown to inhibit apo- induced in LCLs by incubating 5 10 cells/well with 100 ptosis and to regulate lymphoid migratory pathways ng/ml of anti-Fas monoclonal antibody CH-11 (IgM; Medical and Biological Laboratories, Watertown, MA) in 96-well mi- (17,18). croplates. Control mouse IgM monoclonal antibody (TEPC- 183; Sigma, St. Louis, MO) was used in equivalent concentra- PATIENTS AND METHODS tions. Cell death was determined at different time points, using a commercial MTS kit (Promega, Madison, WI). Microplate Study subjects. A panel of LCLs from a total of 78 wells were read using an enzyme-linked immunosorbent assay individuals (23 patients with RA, 8 with systemic lupus ery- plate reader at 490 nm. 756 PI ET AL Analysis of DNA fragmentation. Demonstration of monoclonal anti–caspase 3 (clone 19, IgG2a; Signal Transduc- Ј DNA laddering on agarose gels was performed as previously tion Lab, Lexington, KY) followed by sheep F(ab )2 fragment described (20), with some modifications. Briefly, cells were horseradish peroxidase–conjugated, anti-mouse immunoglob- lysed in Tris–EDTA (TE) buffer containing 0.2% Triton ulin (Amersham, Piscataway, NJ). For poly(ADP-ribose) poly- X-100, pH 8.0. Fragmented DNA was separated from intact merase (PARP) immunoblots, cell lysates (10 ␮g of protein per chromatin by microfuging at 14,000 rpm at 4°C for 20 minutes. lane) were separated on 7.5% SDS–polyacrylamide gels, trans- The resulting supernatant was treated with 1 mg/ml of protein- ferred to PVDF membranes, and probed with monoclonal ase K at 37°C overnight, then extracted with phenol– mouse anti-human PARP antibody C2.10 (IgG1; Enzyme chloroform–isoamyl alcohol (at a ratio of 25:24:1) 3 times.
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