The objective of this study was to characterize the expression of interleukin-1 receptor antagonist (IL-1rn) in immunodepleted murine mesenchymal stem cells (IDmMSCs). To better characterize IDmMSCs, we also catalogued their transcriptome via serial analysis of gene expression (SAGE). Interrogation of the SAGE database revealed an abundance of SAGE tags corresponding specifically to mRNA encoding IL-1rn, a potent anti-inflammatory protein that antagonizes the activity of interleukin-1 (IL-1). Expression of IL-1rn by IDmMSCs may explain, in part, their immunomodulatory activity and ability to ameliorate various injuries. Consequently, we characterized the expression of IL-1rn in IDmMSCs and evaluated its biological activity using a T-cell proliferation assay. Screening an IDmMSC cDNA library by PCR confirmed that the cells expressed transcripts corresponding to IL-1rn, thereby validating the SAGE data. To directly evaluate protein expression, IDmMSCs were isolated from bone marrow collected from the long bones of FVB/n mice via immunodepletion. Briefly, whole bone marrow cells were cultured in a-MEM supplemented with 10% FBS in a humidified chamber with 5 % CO2 at 378C for 4 days, fed with fresh media, and incubated an additional 3-5 days. Cells were then immunodepleted by incubation with M-280 Dynabeads conjugated to anti-CD11b, CD34, and CD45 antibodies. Enzyme-linked immunosorbent assays (ELISA) quantitatively demonstrated that IDmMSCs secreted high levels of IL-1rn protein, 1ng/mL/cell (p < .01). Immunofluorescence staining further revealed that IL-1rn expression was restricted to specific subpopulations of IDmMSCs, and FACS analysis confirmed that approximately 24% of IDmMSCs expressed the protein. An in vitro T-cell proliferation assay was employed to demonstrate that IL-1rn secreted by IDmMSCs inhibited the ability of IL-1 to induce proliferation of the T cells (p < .01). 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 imply 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.
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