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251 ENDOGENOUS EXPRESSION OF INTERLEUKIN-1 RECEPTOR ANTAGONIST ISOFORMS BY MURINE MESENCHYMAL STEM CELLS.
  1. A. C. Pandey1,
  2. M. F. Dutreil1,
  3. D. G. Phinney1
  1. 1Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, LA

Abstract

Mesenchymal stem cells (MSCs) are pluripotent cells derived from the bone marrow possessing a very high potential to regulate the cellular microenvironment due to expression of a vast array of cytokines, chemokines, and adhesion factors. The objective of this study was to characterize the expression and biological activity of interleukin-1 receptor antagonist (IL-1rn) in immunodepleted murine mesenchymal stem cells (IDmMSCs). We hypothesized that expression of IL-1rn by IDmMSCs may explain, in part, their immunomodulatory activity and ability to ameliorate various injuries/disease states. IL-1rn production of the IDmMSCs was compared with a multipotent murine MSC line, CRL-12424. Screening of an IDmMSC cDNA library by PCR confirmed expression of transcripts corresponding to IL-1rn. To directly evaluate protein expression, IDmMSCs were isolated from bone marrow collected from the long bones of FVB/n mice via immunodepletion. Enzyme-linked immunosorbent assays (ELISAs) quantitatively demonstrated that IDmMSCs produced high levels of the secreted isoform of IL-1rn (sIL-1rn). Production of sIL-1rn in IDmMSCs was fivefold higher than the CRL-12424 cells, with the average secretion of the IDmMSCs totaling 1 ng/mL/cell. Immunofluorescence staining further revealed that intracellular IL-1rn (icIL-1rn) expression was restricted to specific subpopulations of IDmMSCs, and FACS analysis confirmed that 24% of IDmMSCs expressed the protein. Interestingly, FACS analysis of the depleted cell populations demonstrated no expression of icIL-1rn, indicating that expression is specific and limited to the IDmMSCs subpopulation. An in vitro T-cell proliferation assay was employed to demonstrate that conditioned medium from IDmMSCs, containing IL-1rn, inhibited the ability of IL-1α to induce proliferation of the T cells. The results of the assay showed that sIL-1rn in the conditioned media of IDmMSCs completely inhibited T-cell proliferation. Collectively, these results demonstrate that subpopulations of IDmMSCs express high levels of IL-1rn and that this protein can inhibit T-cell proliferation. By counteracting the effects of IL-1 in vivo, IL-1rn may regulate T-cell proliferation and bone metabolism. Additionally, these findings implicate that IL-1rn expression contributes to the immunomodulatory effects of MSCs, as demonstrated in the amelioration of various injuries such as bleomycin-induced lung injury, perhaps via immunomodulation of the cellular microenvironment.

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