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.

apoptosis - autoimmunity - CD95 - human - T Cells

Apoptose - Autoimmunität - CD95 - Human - T-Zellen

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

Correspondence