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THE STEM CELL NICHE

Multilevel Regulation of IL-6R by IL-6–sIL-6R Fusion Protein According to the Primitiveness of Peripheral Blood-Derived CD133⁺ Cells

^a Laboratoire Micro-Environnement et le Renouvellement Cellulaire Intégré (EA 3829), Faculté de Médecine-Pharmacie de Rouen, Rouen, France;
^b Department of Biochemistry, Christian-Albrechts-Universität zu Kiel, Kiel, Germany

Key Words. CD133⁺ cells • Hyperinterleukin-6 • IL-6R • ADAM proteases • 5-Fluorouracil

Correspondence: David Campard, Ph.D., Laboratoire Micro-Environnement et le Renouvellement Cellulaire Intégré (MERCI), Faculté de Médecine-Pharmacie, 22 boulevard Gambetta, 76183 Rouen cedex, France. Telephone: 33-235-148350; Fax: 33-235-148340; e-mail: david.campard{at}etu.univ-rouen.fr

Received April 15, 2005; accepted for publication December 7, 2005.
Interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are major factors for maintenance and expansion of hematopoietic stem cells (HSCs). Sensitivity of HSCs to IL-6 has been previously studied, in part by measuring the expression of IL-6R on the membrane (mIL-6R). Several studies have described the regulation of cell surface expression of IL-6R by several cytokines, but the role of glycoprotein 130 activation has not yet been investigated. In this study, CD133⁺ cells were purified from adult peripheral blood and were precultured in the absence or presence of 5-fluorouracil (5-FU) for selection of quiescent HSCs. Cells were cultured with continuous or pulsed stimulations of an IL-6 –sIL-6R fusion protein (hyperinterleukin-6 [HIL-6]) to 1) detect mIL-6R by flow cytometry, 2) assess mIL-6R and sIL-6R RNAs by reverse transcription-polymerase chain reaction, 3) measure sIL-6R in supernatants by enzyme-linked immunosorbent assay, 4) analyze cell-cycle status, and 5) perform long-term culture-initiating cell assays. The level of mIL-6R^– cells was preserved by 5-FU incubation. HIL-6 increased steady-state mIL-6R RNA and expression rate on HSCs, independently of treatment with 5-FU. Enhanced production of sIL-6R was observed with short pulses of HIL-6 on CD133⁺ 5-FU-pretreated cells. This overproduction of sIL-6R was abrogated by tumor necrosis factor- protease inhibitor-1, an inhibitor of a disintegrin and metalloprotease proteases, suggesting the shedding of mIL-6R. This phenomenon was mediated through the phosphatidylinositol-3'-kinase pathway and was involved in the maintenance of primitive HSCs. In conclusion, expression and production of IL-6R are tightly regulated and stage specific. We assume that sIL-6R produced by shedding should be involved in autocrine and paracrine loops in the HSC microenvironment.

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