PMC

Levels of pulmonary cytokines (pg/ml) in multiplex assays of lung homogenates (mean ± SD, with 5–10 animals per group) in mice expressing IL-33 isoforms as indicated.

ELISA for mIL-33 of TC-1 cell culture supernates (open bars) and lysates (shaded bars), in triplicate, 48 h after infection with AdV constructs encoding the indicated proteins. The same ELISAs were repeated in A549 and SAEC cells with similar results.

Histological changes in mouse lungs following flmIL-33 gene delivery 14 days after AdV construct instillation. Shown from top to bottom are H&E staining of lung sections from mice infected with viral construct for delivery of indicated IL-33 isoforms; immunohistochemistry of flmIL-33-expressing mouse lungs for CD3, B220, Mac3, and Ly6G, as indicated; periodic acid-Schiff staining of flmIL-33- (lower left) and mmIL-33-expressing (lower right) mouse lungs.

Levels of IL-33 in nuclear (closed bars) and cytoplasmic (open bars) fractions of TC-1 cells infected with AdV encoding the indicated proteins (pg per μg total protein ± SD) measured by ELISA. Below, western blotting of the same nuclear (N) and cytoplasmic (C) samples for histone deacetylase 2 (HDAC2) and β-actin.

Responses to IL-33 gene delivery in ST2-deficient animals. The top two panels show BAL cell counts in flmIL-33- and mmIL-33-expressing mice on day 14 after construct instillation in wild-type (left) and ST2 −/− (right) mice. Significant differences from AdV-NULL-challenged mice are indicated with asterisks (p < 0.05). Below, histological changes in the lungs following flmIL-33 or mmIL-33 gene delivery (H&E staining).

Changes in cellular composition of bronchoalveolar lavage induced by gene delivery of IL-33 to the lungs of C57Bl/6 mice in vivo (mean ± SD), with 3–7 animals per group and with significant differences from AdV-NULL-challenged mice indicated with asterisks (p < 0.05). The top panel shows BAL cell counts in mice overexpressing the indicated IL-33 isoforms on day 14. The bottom panel shows the dynamics of BAL lymphocytes and neutrophils in flmIL-33-overexpressing mice.

Regulation of gene expression by flmIL-33 and mmIL-33 in cell culture (A) and in vivo (B). A. Mean fold increase ± SD in the levels of indicated mRNAs in TC-1 cells infected with indicated constructs versus cells infected with AdV-NULL, 48 h after infection, by RT-Q-PCR. Repeated on two occasions with similar results. Note the log scale on the vertical axis. B. Western blot of mouse lung homogenates for MMP10 and β-actin, 14 days after instillation of adenoviruses encoding IL-33 isoforms. Sample loading was normalized to the entire left lung: the whole lung was homogenized and an equal fraction of the homogenate loaded on the gel in each case.

Validation of infectivity and IL-33 gene delivery by recombinant AdV constructs. A. Recombinant adenoviruses infect cells causing green fluorescence due to GFP expression encoded in the viral backbone. Bright field microscopy (left), GFP fluorescence microscopy (middle), and image overlay (right) of AdV-flmIL-33-infected mouse epithelial TC-1 cells 48 h after infection (×20 objective). Similar results were obtained with all other constructs in these cells and also in A549 primary small airway epithelial cells and primary pulmonary mouse fibroblasts. No fluorescence was observed in cells without AdV infection. B. Western blotting with anti-mIL-33 antibody of fibroblast culture lysates infected with AdV encoding the indicated proteins. rmIL-33 is a commercial preparation of mature IL-33 (R&D Systems). Samples were normalized to total protein for loading. C. ELISA of whole lung homogenates for GFP, ng/ml ± SD, on day 14 after instillation of adenoviruses encoding the indicated proteins. D. ELISA of whole lung homogenates for mIL-33, ng/ml ± SD. WT mice (open bars) and ST2−/− mice (closed bars) were analyzed 14 days after infection with AdV encoding the indicated proteins, with 3–5 animals per group.

Expression of IL-33 in human lung. A-H. Immunohistochemistry for IL-33 in lung tissue obtained from one healthy volunteer and three patients with idiopathic pulmonary fibrosis (IPF), as indicated. Similar observations were obtained in two additional healthy controls and three additional patients with IPF. This anti-IL-33 antibody (Ab) indiscriminately reacts with full-length and mature forms of hIL-33 (brown staining). Lung tissue stained with isotype control Ab (A), or with anti-IL-33 Ab from the healthy control (B) and 3 patients with IPF were imaged with a ×10 objective (C, E). Selected areas were imaged with a ×20 objective (D, F) or a ×40 objective (G, H). I. ELISA for total IL-33 in BAL from five healthy volunteers (light circles) and ten patients with IPF (dark circles). J. Western blotting of lung tissue homogenates from four patients with IPF (IPF) and two healthy controls (Ctrl) for IL-33 (upper gel) and β-actin (lower gel). Recombinant mature human (mh) IL-33 protein was used as a positive control in the right-most column. Loading of the samples was normalized to total protein.