Figure 3

(a) Representative views of airway mucosa from four lungs double-stained for protein gene product 9.5 (PGP9.5, green) and gastrin releasing peptide (GRP, red). The upper panel is the lumen view, the middle one is rotated through 30 deg and the lower one through 90 deg, ie view from the side. Strings of varicose nerves are present in the epithelium. The dark holes indicate the location of goblet cells. PNEC are inconspicuous in the upper view but become more apparent when the field is projected at an angle. The lower panels demonstrate that GRP is present predominantly in the PNEC processes, whereas PGP 9.5 is restricted to the cell body with its apical stem. Nerves feature strongly in the apical epithelium. Bar = 50μm. Boxed area: This PNEC has been turned through 90 degrees so that the prominent processes now point upwards, and enlarged (right, upper panel). A 90 deg rotation of the projection reveals that the PNEC processes stain strongly for GRP whereas the cell body and apical stem stain mainly for PGP 9.5. Some of the processes are lumen-directed, other processes with feet-like appearance are directed toward the lamina propria. Bar = 10μm. (b) Three PNEC in a field from another lung shown from the lumen (upper) and rotated through 90 deg (lower). Two of the PNEC are predominantly GRP positive whereas the third PNEC stains strongly for PGP 9.5. The upper stem of the middle cell body stains yellow indicating that the PGP 9.5 and the GRP staining are about equal. Bar = 10 μm. (c) A single PNEC shown as individual fields: PGP9.5 only (left), GRP only (middle), composite PGP9.5 + GRP (right). GRP reveals fine processes that issue from the cell body. In contrast to PGP 9.5, GRP does not stain the cell nucleus. Bar = 10 μm. (d) A single PNEC in close association with a nerve terminal. A nerve rises from the base of the PNEC, climbs through the epithelium along the PNEC stem and spreads laterally in the apical epithelium where it exhibits enlarged terminal varicosities. Upper panel: lumen view, middle panel: 45 deg rotation, lower panel, 90 degree rotation. Bar 10 μm. (e) Lumen view of mucosa where the epithelium is tilted showing three PNEC from an angle. Patches of fine varicose nerves are present. Some of the nerves lie close to the stems of two of the PNEC (arrow heads). Bar = 25 μm. Boxed area(right): High power view after rotating shows two PNEC within a patch of nerves. The left PNEC is strongly PGP9.5 positive. The right PNEC has several processes in close apposition to nerves that rise through the epithelium to form an apical nerve plexus. Nerves in the apical epithelium lie close to the central stems of both PNEC. Bar = 10 μm.