FIGURE 5.

Biomechanical strain induces IL-33 secretion from living cells. A, biomechanical strain induces IL-33 secretion. Fibroblasts stably expressing TC-IL-33 were subjected to 1 Hz cyclic 8% biaxial stretch for the number of hours indicated. Extracellular media was assayed via ELISA, and raw optical density values converted to IL-33 concentration via a standard curve followed by normalization to IL-33 in total cellular lysate. At 4 h, a 3.9-fold increase in optical density was measured (p = 0.012 by ANOVA; *, p < 0.01 for 0 versus 4 h by Bonferroni post-test; n = 4 per time point; whiskers represent mean and S.E.). B, IL-33 is released in full-length form. TC-IL-33 expressing cells were subjected to cyclic biaxial stretch for 4 h or left unstrained as control. Culture media was sampled, and TC-IL-33 was captured in a microplate coated with an anti-C-terminal IL-33 antibody. N-terminal tetracysteine fluorescence was induced by exposure to resorufin derivative dye. Raw optical density was normalized to the signal from total cellular lysate. A 1.8-fold increase in fluorescence was noted under conditions of strain compared with control (*, p = 0.008; n = 4 per condition; whiskers represent mean and S.E.). C, IL-33 is released from primary cells upon application of mechanical strain. Primary human skin fibroblasts were subjected to cyclic biaxial stretch for 4 h or left unstrained as control. Extracellular media was assayed via ELISA, and raw optical density values converted to IL-33 concentration via a standard curve followed by normalization to IL-33 in total cellular lysate. A 3.0-fold increase in optical density was noted in the context of strain (*, p = 0.02, n = 4–5 per condition; whiskers represent mean and S.E.). D, IL-33 is lost from cells subjected to biomechanical overload in vivo. Lentiviral particles housing HA-IL-33 and a non-fusion GFP reporter were injected into the left ventricle of anesthetized mice. Seven days later, mice underwent sham surgery (upper panels) or 2-h transaortic constriction (TAC, lower panels) to induce acute pressure overload of the left ventricle. Cardiac sections of sham hearts revealed strong HA-IL-33 staining (probed via an anti-HA antibody) in infected cells demarked by GFP expression. In hearts subjected to TAC, HA-IL-33 immunofluorescence was absent from GFP positive cells. Images are representative of 2–3 animals per group and 6–10 microscopic fields per animal. Scale bars represent 10 μm.