Impaired CD95-mediated Apoptosis in Autoimmunity and Occurrence of a p22 Caspase-8 Cleavage Product in JIA

Abstract

Background: Altered apoptosis can lead to autoimmune diseases such as systemic lupus erythematosus (SLE). Juvenile idiopathic arthritis (JIA) is another autoimmune disease characterized by autoinflammation. During this process activated T-cells accumulate in the synovial fluid. We hypothesized that resistance to CD95-mediated apoptosis could contribute to autoimmune phenotypes.

Patients/Method: We isolated highly activated T cells (CD45RO⁺ and CD95⁺) by magnetic separation from healthy controls, JIA patients and patients with other autoimmune diseases. In these purified cells, apoptosis was induced by stimulation with recombinant human soluble CD95 ligand (rhsCD95L) or dexamethasone and analyzed by flow cytometry. In addition, cleavage and expression of apoptosis mediators (Caspase-8 and –3) and regulators (FLIP, Bcl-2, Bcl-x_L) were analyzed in mononuclear cells using immunoblot technique.

Results: Apoptosis upon CD95 stimulation, but not dexamethasone treatment, was reduced in JIA patients and patients with other autoimmune diseases compared to healthy controls. Additionally we observed a non-canonical cleavage pattern of Caspase-8 resulting in a p22 fragment and high expression of FLIP in SFMCs of patients with JIA.

Conclusion: Expression and cleavage of proteins of the CD95 pathway is altered in JIA providing a possible explanation for resistance against death receptor-mediated apoptosis. Dexamethasone-induced apoptosis, however, is intact arguing against a general defect in apoptosis. The implications of the p22 fragment regarding apoptosis have to be further analyzed. The strong expression of FLIP_Short in SFMCs may as well result from the highly activated status of the cells or be a feature of autoimmunity.

Zusammenfassung

Hintergrund: Veränderte Apoptose kann zu Autoimmunerkrankungen wie z.B. Systemischer Lupus Erythematosus (SLE) führen. Eine weitere ist die Juvenile Idiopathische Arthritis (JIA), deren Hauptmerkmal die Autoinflammation ist. Hierbei akkumulieren hochgradig aktivierte T-Zellen in der Synovialflüssigkeit (SF). Wir stellen die Hypothese auf, dass Resistenz gegenüber CD95-vermittelter Apoptose zu Autoimmunität führen kann.

Patienten/Methoden: Das Apoptoseverhalten von mittels Magnetbeads isolierten hochgradig aktivierten T-Zellen (CD45RO⁺ und CD95⁺) von gesunden Kontrollen, JIA-Patienten und Patienten mit anderen Autoimmunerkrankungen wurden nach In-vitro/ex-vivo-Stimulation mit rekombinanten humanen löslichen CD95-Liganden (rhsFasL) und Dexamethason durchflusszytometrisch analysiert. Zusätzlich wurden die Spaltung und die Expression von Mediatoren (Caspase-8 und -3) und Regulatoren (FLIP; bcl-2;bcl-xl) der CD95-induzierten Apoptose in mononukleären Zellen (MNCs) mittels Immunoblot analysiert.

Resultate: Im Vergleich zu gesunden Kontrollen war die Apoptose nach CD95-Stimulation, jedoch nicht nach Dexamethason-Stimulation bei JIA und anderen Autoimmunerkrankungen reduziert. Wir konnten zusätzlich bei JIA eine alterierte, in einem p22-Fragment resultierende, Caspase-8-Spaltung zeigen sowie eine erhöhte FLIP_Short-Expression in SFMCs.

Schlussfolgerung: Expression und Spaltung von Proteinen der CD95-induzierten Apoptose ist bei JIA verändert, was eine mögliche Erklärung für eine unvollständige, Todesrezeptor-vermittelte Apoptose sein könnte. Dexamethason-induzierte Apoptose ist in vitro intakt, was gegen einen generellen Apoptosedefekt spricht. Um die Auswirkung des p22-Fragments auf die Apoptose weiter zu klären, sind weitergehende Untersuchungen notwendig. Die starke Expression von FLIP_Short in den SFMCs kann sowohl durch den hohen Zellaktivierungsgrad entstanden, als auch ein Resultat der Autoimmunität sein.

Key words

apoptosis - autoimmunity - CD95 - human - T Cells

Schlüsselwörter

Apoptose - Autoimmunität - CD95 - Human - T-Zellen

Literatur

• 1 Barnhart BC, Alappat EC, Peter ME. The CD95 type I/type II model.  Semin Immunol. 2003;  15 185-193
• 2 Borchers AT, Selmi C, Cheema G. et al . Juvenile idiopathic arthritis.  Autoimmun Rev. 2006;  5 279-298
• 3 Budd RC, Yeh WC, Tschopp J. cFLIP regulation of lymphocyte activation and development.  Nat Rev Immunol. 2006;  6 196-204
• 4 Catrina AI, Ulfgren AK, Lindblad S. et al . Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium.  Ann Rheum Dis. 2002;  61 934-936
• 5 Cleary AG, Sills JA, Davidson JE. Revision of the proposed classification criteria for juvenile idiopathic arthritis: Durban, 1997.  J Rheumatol. 2000;  27 1568
• 6 Dutton A, Young LS, Murray PG. The role of cellular FLICE inhibitory protein (c-FLIP) in the pathogenesis and treatment of cancer.  Expert Opin Ther Targets. 2006;  10 27-35
• 7 Holzelova E, Vonarbourg C, Stolzenberg MC. et al . Autoimmune lymphoproliferative syndrome with somatic Fas mutations.  N Engl J Med. 2004;  351 1409-1418
• 8 Knipp S, Feyen O, Ndagijimana J. et al . Ex vivo apoptosis, CD95 and CD28 expression in T cells of children with juvenile idiopathic arthritis.  Rheumatol Int. 2003;  23 112-115
• 9 Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology.  Cell. 2001;  104 487-501
• 10 Massa M, De Benedetti F, Pignatti P. et al . Anti-double stranded DNA, anti-histone, and anti-nucleosome IgG reactivities in children with systemic lupus erythematosus.  Clin Exp Rheumatol. 1994;  12 219-225
• 11 Medema JP, Scaffidi C, Kischkel FC. et al . FLICE is activated by association with the CD95 death-inducing signaling complex (DISC).  EMBO J. 1997;  16 2794-2804
• 12 Niehues T, Feyen O, Telieps T. Concepts on the pathogenesis of juvenile idiopathic arthritis.  Z Rheumatol. 2008;  67 111-116
• 13 Peter ME, Krammer PH. The CD95(APO-1/Fas) DISC and beyond.  Cell Death Differ. 2003;  10 26-35
• 14 Puck JM, Straus SE. Somatic mutations–not just for cancer anymore.  N Engl J Med. 2004;  351 1388-1390
• 15 Rébé C, Cathelin S, Launay S. et al . Caspase-/8 prevents sustained activation of NF-kappaB in monocytes undergoing macrophagic differentiation.  Blood. 2007;  109 1442-1450
• 16 Sartorius U, Schmitz I, Krammer PH. Molecular mechanisms of death-receptor-mediated apoptosis.  Chembiochem. 2001;  2 20-29
• 17 Saurenmann RK, Levin AV, Feldman BM. et al . Prevalence, risk factors, and outcome of uveitis in juvenile idiopathic arthritis: a long-term followup study.  Arthritis Rheum. 2007;  56 647-657
• 18 Schmitz I, Weyd H, Krueger A. et al . Resistance of short term activated T cells to CD95-mediated apoptosis correlates with de novo protein synthesis of c-FLIP_short.  J Immunol. 2004;  172 2194-2200
• 19 Seidi OA, Sharief MK. The expression of apoptosis-regulatory proteins in B lymphocytes from patients with multiple sclerosis.  J Neuroimmunol. 2002;  130 202-210
• 20 Smolewska E, Stanczyk J, Robak T. et al . Inhibited apoptosis of synovial fluid lymphocytes in children with juvenile idiopathic arthritis is associated with increased expression of myeloid cell leukemia 1 and XIAP proteins.  J Rheumatol. 2006;  33 1684-1690
• 21 Ueffing N, Schuster M, Keil E. et al . Up-regulation of c-FLIP short by NFAT contributes to apoptosis resistance of short-term activated T cells.  Blood. 2008;  112 690-698
• 22 Villanueva J, Lee S, Giannini EH. et al . Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome.  Arthritis Res Ther. 2005;  7 R30-R37
• 23 Vutova P, Wirth M, Hippe D. et al . Toxoplasma gondii inhibits Fas/CD95-triggered cell death by inducing aberrant processing and degradation of caspase 8.  Cell Microbiol. 2007;  9 1556-1570

1 The authors contributed equally to this work and should be considered co-first authors.

Correspondence