Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury

Abstract

Mesenchymal stem cells (MSCs) have been exploited as cellular vectors to treat a wide array of diseases but the mechanisms responsible for their therapeutic effect remain indeterminate. Previously, we reported that MSCs inhibit bleomycin (BLM)-induced inflammation and fibrosis within the lungs of mice. Interrogation of the MSC transcriptome identified interleukin 1 receptor antagonist (IL1RN) as a potential mediator of this effect. Fractionation studies indicated that MSCs are the principal source of IL1RN in murine bone marrow and that its expression is restricted to a unique subpopulation of cells. Moreover, MSC-conditioned media was shown to block proliferation of an IL-1alpha-dependent T cell line and inhibit production of TNF-alpha by activated macrophages in vitro. Studies conducted in mice revealed that MSC administration was more effective than recombinant IL1RN delivered via adenoviral infection or osmotic pumps in inhibiting BLM-induced increases in TNF-alpha, IL-1alpha, and IL1RN mRNA in lung, IL1RN protein in bronchoalveolar lavage (BAL) fluid, and trafficking of lymphocytes and neutrophils into the lung. Therefore, MSCs protect lung tissue from BLM-induced injury by blocking TNF-alpha and IL-1, two fundamental proinflammatory cytokines in lung. Identification of IL1RN-expressing human MSC subpopulations may provide a novel cellular vector for treating chronic inflammatory diseases in humans.

Fig. 1.

Murine MSCs express IL1RN mRNA and protein. (a) Screening of a murine MSC cDNA library by PCR yielded a 338-bp fragment corresponding to IL1RN mRNA. (b) Levels of secreted IL1RN protein were quantified by ELISA on a per-cell basis from unfractionated plastic adherent marrow cells (predepleted) or murine MSCs enriched by immunodepleted. Plotted values (mean ± SD) represent duplicates from four separate experiments. ∗, P < 0.0007.

Fig. 2.

Distribution of IL1RN in murine bone marrow. (a–e) FACS analysis revealed that CD11b (a) and CD45 (c) positive cells recovered from magnetic beads after immunodepletion lacked expression of IL1RN protein (b and d), but 24% of immunodepleted murine MSCs expressed IL1RN (e). (f) Immunostaining of murine MSCs with an anti-IL1RN antibody (red) and DAPI (blue) confirmed that IL1RN expression was restricted to a specific subpopulation of cells.

Fig. 3.

IL1RN secreted by MSCs antagonizes IL-1α activity and blocks release of TNF-α from activated macrophages. (a) Equivalent numbers of D10.G4.1 cells were cultured for 5 days in media alone or media supplemented with the indicated concentrations of IL-1α, recombinant IL1RN (rIL1RN), MSC-conditioned media (CM), or a neutralizing IL1RN antibody (anti-IL1RN), and then the total yield of cells for each condition was determined by counting. ∗, P < 1 × 10⁻¹⁰; ∗∗, P < 0.005. (b and c) Levels of TNF-α secreted by the macrophage cell line RAW-264.7 after exposure to silica (b) or LPS (c) in the presence or absence of MSC-conditioned media (≈3 ng of IL1RN) was quantified by ELISA. ∗, P < 0.000005; ∗∗, P < 0.0005; #, P < 0.001. Plotted values (mean ± SD) represent duplicates from three separate experiments.

Fig. 4.

MSC administration alters BLM-induced inflammatory responses in lung. BAL fluid harvested from mice 3 days after BLM challenge was analyzed for total cell number (a), cell composition (b), and levels of IL1RN protein (c). Total RNA harvested from lung tissue was analyzed by real-time PCR to quantify TNF-α mRNA levels (d). Reported values represent the average ± SD (bars) calculated from animals within each treatment group. ∗, P < 0.05; ∗∗, P < 0.01; #, P < 0.005; n = 5 mice per group.

Fig. 5.

MSC administration inhibits BLM-induced up-regulation of IL-1α and IL1RN mRNA in lung. Relative expression levels of IL-1α (a) and IL1RN (b) mRNA in lung at 7 and 14 days after BLM exposure was quantified by real-time PCR, using the relative Ct method. Plotted values represent the average ± SD (bars) calculated from animals within each treatment group and then normalized to 7 day saline control treated animals (n = 3–7 mice). ∗, P < 0.0002; ∗∗, P < 0.01; #, P < 0.05.

Fig. 6.

IL1RN-expressing human MSCs. Photomicrographs of human MSCs immuno-stained with an anti-IL1RN antibody (green) and DAPI (blue). (Magnifications: a, ×200; b, ×400) IL1RN-expressing and nonexpressing MSCs are demarked by arrows and arrow heads, respectively. (c) FACS analysis of human MSCs revealed that ≈5% of cells (R2 gate) expressed IL1RN protein.