ENDOGENOUS EXPRESSION OF INTERLEUKIN-1 RECEPTOR ANTAGONIST ISOFORMS BY MURINE MESENCHYMAL STEM CELLS.

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.