The idiopathic pulmonary fibrosis honeycomb cyst contains a mucocilary pseudostratified epithelium

Abstract

Background: We previously identified a MUC5B gene promoter-variant that is a risk allele for sporadic and familial Idiopathic Pulmonary Fibrosis/Usual Interstitial Pneumonia (IPF/UIP). This allele was strongly associated with increased MUC5B gene expression in lung tissue from unaffected subjects. Despite the strong association of this airway epithelial marker with disease, little is known of mucin expressing structures or of airway involvement in IPF/UIP.

Methods: Immunofluorescence was used to subtype mucus cells according to MUC5B and MUC5AC expression and to identify ciliated, basal, and alveolar type II (ATII) cells in tissue sections from control and IPF/UIP subjects. Staining patterns were quantified for distal airways (Control and IPF/UIP) and in honeycomb cysts (HC).

Results: MUC5B-expressing cells (EC) were detected in the majority of control distal airways. MUC5AC-EC were identified in half of these airways and only in airways that contained MUC5B-EC. The frequency of MUC5B+ and MUC5AC+ distal airways was increased in IPF/UIP subjects. MUC5B-EC were the dominant mucus cell type in the HC epithelium. The distal airway epithelium from control and IPF/UIP subjects and HC was populated by basal and ciliated cells. Most honeycombing regions were distinct from ATII hyperplasic regions. ATII cells were undetectable in the overwhelming majority of HC.

Conclusions: The distal airway contains a pseudostratified mucocilary epithelium that is defined by basal epithelial cells and mucus cells that express MUC5B predominantly. These data suggest that the HC is derived from the distal airway.

Figure 1. MUC5B is the dominant gel-forming mucin in the normal distal airway epithelium.

A–D: Hematoxylin and eosin (H&E) staining of the normal conducting airway. A. Proximal airway; B–D. Distal airways. L, lumen; Arrow: cartilage; Arrowheads: black -submucosal gland, yellow - airway epithelium, blue - smooth muscle bands. Final magnification: 10× E–L: DIF analysis of MUC5B (red), MUC5AC (green) and nuclei (blue). E. Proximal airway. L: lumen; arrow: submucosal gland. F–H. Distal airways. Panels A & E; B & F; C & G; and D & H are adjacent sections. Representative images of airways expressing both MUC5B and MUC5AC (B, F); only MUC5B (C, G); neither MUC5B nor MUC5AC (D, H). Values in each panel indicate the frequency with which the pattern was detected. Final magnification, 10× I–L Single color images of the region indicated in panel F. Final magnification, 20×. M: Pie chart indicating the frequency of distal airways expressing MUC5B and/or MUC5AC. N: Frequency distribution of MUC5B (red) and MUC5AC (green) in distal airways. Asterisks represent significant p value (<0.05) obtained for test of proportions of that category.

Figure 2. MUC5B and MUC5AC expression is increased in IPF/UIP distal airways.

A–B: Hematoxylin and eosin (H&E) staining of the IPF/UIP distal airways. Asterisk-airway lumen; arrowhead - epithelium. Final magnification: 10× C-D: DIF analysis of MUC5B (red), MUC5AC (green) and nuclei (blue). Panels A & C; B & D are adjacent sections. Values in each panel indicate the frequency with which the pattern was detected. Final magnification,10× E-F: Pie charts indicating the frequency of distal airways expressing MUC5B and/or MUC5AC in normal subjects (E, represented from Fig 1N for comparison purposes) and IPF/UIP subjects (F). G: Frequency of MUC5B+distal airways in control and IPF/UIP patients. H. Frequency distribution of MUC5B-expressing cells in control (red, represented from Fig 1N for comparison purposes) and IPF/UIP (red-checked) subjects. I: Frequency of MUC5AC+distal airways in control and IPF/UIP patients. J. Frequency distribution of MUC5AC-expressing cells in control (green, represented from Fig 1N for comparison purposes) and IPF/UIP (green-checked) subjects. Asterisks in Fig 1H,J represent significant p value (<0.05) obtained for test of proportions of that category.

Figure 3. MUC5B is the dominant gel-forming mucin in the honeycombing cyst.

A-B: Hematoxylin and eosin (H&E) staining of the honeycombing cyst. The region indicated in panel A (10×) is shown at higher magnification in B (20×). Arrows: yellow -pseudostratified epithelium, green - simple epithelium, red - disrupted epithelium, blue -fibrosis. Asterisk-mucus plug. C-H: DIF analysis of MUC5B (red), MUC5AC (green) and nuclei (blue). The region indicated in panels C (10×) is shown at higher magnification in panel D. D: Arrows: yellow -pseudostratified epithelium, green - simple epithelium, red - disrupted epithelium. The region indicated in panel E (10×) is shown at higher magnification in F-H (20×). Arrows: yellow MUC5B/MUC5AC dual-positive cells, red-MUC5B single-positive cell.

Figure 4. The honeycomb cyst contains a ciliated epithelium.

A–F: DIF analysis of MUC5B (red), acetylated tubulin (ACT, green), and nuclei (blue). Control distal airway, A (10×) and B (20×). IPF/UIP distal airway, C (10×) and D (20×). Arrows: green - ciliated cells in the mucosecretory pseudostratified epithelium. Honeycomb cyst: E: (10×), F (20×), G-H (40×). Asterisks, mucus plug; HC, honeycomb cyst. Arrows: green - ciliated cells in the mucosecretory pseudostratified epithelium, red - MUC5B+cells in the mucosecretory pseudostratified epithelium, yellow - ciliated cells in the pseudostratified epithelium, blue - ciliated cells in the simple epithelium.

Figure 5. Basal cells populate the pseudostratified regions of the HC epithelium.

A-F: DIF analysis of Keratin 5 (K5, red), MUC5B (green) and nuclei (blue). Control distal airway, A (10×) and B (20×). IPF/UIP distal airway, C (10×) and D (20×). Honeycomb cyst, E (10×) and F (20×). Asterisk, mucus plug; HC, honeycomb cyst. Double arrows: Auto-fluorescent red blood cells and elastin bands. Arrows: red - K5+cells; green - MUC5B+cells. G: DIF analysis of K5 and K14 in honeycomb cyst, G (10×). The region indicated in panel G is shown at higher magnification in G1 (20×). H, I: DIF analysis of K5 and K14 in IPF/UIP distal Airway (40×). Arrows: orange - dysplastic basal cells; pink - K5/K14 dual-positive cells.

Figure 6. Alveolar type II (ATII) cells are absent from the pseudostratified regions of the HC.

A-H: DIF analysis of pro-surfactant protein C (proSPC, red), MUC5B (green) and nuclei (blue). Control lung parenchyma, A (10×) and B (20×). IPF/UIP alveolar type II (ATII) cell hyperplasia, C (10×) and D (20×). IPF/UIP ATII-lined cyst, E (10×) and F (20×). Honeycomb cyst, G (10×) and H (20×). Asterisks, mucus plugs; HC, honeycomb cyst. I: IPF/UIP transitional zone. Arrows: yellow – pseudostratified epithelium. I-J IPF/UIP transitional zone. I: DIF analysis of pro-surfactant protein C (red), MUC5B (green), and nuclei (blue). Arrows: red - limit of the pro-SPC+simple epithelial region; yellow - luminal mucus; white - proSPC/MUC5B double-negative transition zone; green-MUC5B+pseudostratified region. J: DIF analysis of MUC5B (red), ACT (green), and nuclei (blue). Arrows, blue-simple epithelial region; red - MUC5B+cells, green-ACT+cells.