Fig. 1.

Allergen challenge induces ORMDL3 expression in epithelium in vitro and in vivo. (A) WT mice were challenged with OVA allergen by inhalation. RNA was extracted from lungs. qPCR for ORMDL3 was normalized to the housekeeping gene GAPDH. No OVA vs. OVA (P < 0.05). A549 lung epithelial cells incubated with tobacco smoke media (TS) (B) or mouse macrophage cell line RAW 264.7 (C) incubated with LPS for 3 h. RNA was extracted. qPCR for ORMDL3 was normalized to GAPDH. P < 0.05 for No TS vs. TS and No LPS vs. LPS. (D) WT mice were challenged with Alternaria allergen by inhalation. RNA was extracted from bronchial brushing derived epithelial cells. qPCR for ORMDL-1, ORMDL-2, and ORMDL3 was normalized to the housekeeping gene GAPDH. Alternaria allergen challenge induced a 127-fold increase in levels of expression of ORMDL3 mRNA (P < 0.01 Alt vs. naïve). Levels of ORMDL-1 did not change, whereas levels of ORMDL-2 increased (P < 0.05 Alt vs. naïve), but less than that noted with ORMDL3 (Alt ORMDL3 vs. Alt ORMDL-2; P < 0.05). Results are from three separate experiments with four mice per group. Double-label immunofluorescence microscopy (E–J) and double-label confocal immunofluorescence microscopy (H–J) was performed on human lung epithelial cells (A549) with an anti-ORMDL Ab and an anti-calreticulin Ab (detects ER). (K) Lung sections from non-OVA challenged WT mice have low levels of ORMDL immunostaining. In contrast, lung sections from OVA challenged WT mice (L) have increased ORMDL immunostaining particularly in airway epithelium and in peribronchial mononuclear cells as quantified (M) by light microscopy and image analysis (P < 0.005 vs. no OVA). Lungs from collagen 1 GFP reporter mice (N) have fibroblasts that fluoresce green but do not colocalize with the anti-ORMDL Ab (red). (O) Lungs from α-smooth muscle actin RFP reporter mice have smooth muscle that fluoresce red but does not colocalize with the anti-ORMDL Ab (green; white arrowheads points to green ORMDL+ cell, likely macrophage). RNA was extracted from either (P) BAL macrophages, (Q) bone marrow derived eosinophils, or (R) peripheral blood neutrophils. qPCR for ORMDL1, 2, 3 was normalized to the housekeeping gene GAPDH. ORMDL3 is significantly induced in BAL macrophages (P < 0.05, No Alternaria vs. Alternaria) (P), but not in neutrophils (R). Bone marrow eosinophils (Q) express higher levels of ORMDL3 compared to ORMDL2 or ORMDL1 (P < 0.05). qPCR results are from two separate experiments with four mice per group.