Localization of the low-affinity nerve growth factor receptor p75 in human limbal epithelial cells

Abstract

Biological effects of nerve growth factor (NGF) are mediated through receptors known as nerve growth factor receptors (NGFR), which include p75 and TrkA. This study was initiated after identifying NGFR as an up-regulated gene in the limbus by cDNA microarray analysis and we postulate that its expression may be indicative of a stem/progenitor cell phenotype. Immunohistochemistry was performed on normal human adult (n=5) and foetal (n=3) corneal tissue using antibodies directed against p75, TrkA, NGF, p63, ABCG2 and CK3/12. Limbal, conjunctival and pterygium tissue was obtained from patients (n=10) undergoing pterygium resection and used for immunohistochemical assessment. Paraffin-embedded archival human skin specimens (n=4) were also evaluated. In vitro expression of NGFR was determined in limbal, conjunctival and pterygium-derived epithelial cells. p75 was selectively expressed by basal epithelial cells in pterygia, conjunctiva and limbus, but was absent in the central cornea. These results were confirmed with two additional p75 specific antibodies. In contrast, TrkA was found in full-thickness pterygium, conjunctival, limbal and corneal epithelium in both adult and foetal eyes. p75 expression was identified in a small percentage, while TrkA was found on the entire population of cultured conjunctival, limbal and pterygium-derived epithelial cells. This receptor was also observed in selective regions of the human epidermis and hair follicle bulge. Our results illustrate the selective expression of p75 in basal pterygium, conjunctival and limbal epithelium, while staining was absent in adult and foetal central cornea. p75 may represent an additional ocular surface epithelial stem/progenitor cell signature gene.

Fig. 1

Distribution of nerve growth factor receptors (NGFRs) in diseased and normal ocular surface tissue. Pterygium (A–C) and autologous normal bulbar conjunctival (D–F) and limbal (G–I) tissue was serially sectioned and immunohistochemically assessed with an anti-human p75 (clone 7F10; A, D, G and J), an anti-human TrkA (B, E, H and K), or an anti-human NGF (C, F, I and L) antibody. Sections from normal human small intestine (J) and forebrain (K and L) were included as positive controls. Each section was counterstained with haematoxylin to identify cell nuclei and immunoreactivity was denoted by brown cell-associated reactivity. Sections in (A–I) were derived from one patient, with similar results observed in the 10 patients tested. The arrowhead in (A) points to a p75 positive blood vessel and arrows identify suprabasal pterygium epithelial cells with diminished p75 expression. Arrows in (C) and (I) indicate mild NGF reactivity in basal and suprabasal pterygium and limbal epithelium, respectively. Arrowheads in (J) identify a large nerve bundle while arrows signify p75 positive nerve fibres. Arrows in (K) and (L) point to immunoreactive neurons in human forebrain. Original magnification ×400.

Fig. 2

p75 Expression in the Limbus. p75 expression was determined in whole adult corneal specimens (A–C) using three different antibody clones (A, clone 7F10; B, clone 9992 and C, clone NGFR5¹). A similar epithelial cell staining pattern was observed in the conjunctiva (cj), limbus (lm) and peripheral (pc) and central cornea (cc) with each antibody tested. Sections (A, B and D–G) were counterstained with haema-toxylin to identify cell nuclei and immunoreactivity was denoted by brown cell-associated staining. Tissue in part (C) was not counterstained and viewed under fluorescence microscopy. Tissue in A–C was serial sectioned and derived from one representative specimen. Two additional whole eyes were examined and similar results obtained (micrographs not shown). The area encompassed by the squares in A and B;×40 is magnified (insets a¹–a⁴×1000 and b¹; ×400). Original magnification of C–G is ×400. Sections incubated without a primary antibody (inset, b²) or with an isotype control antibody (E; inset) displayed no immunoreactivity. Sections in D–G were derived from normal human small intestine and were incubated with an appropriate p75 antibody (see panel labels). A and B are composed of 3 contiguous micro-graphs. The arrows in A and B identify limbal vasculature; the arrowheads in (a¹ and C) point to diminished p75 expression in suprabasal cells; the asterisks in (a³ and a⁴) depict Bowman's Layer. The central cornea (a⁴) was not captured in the low powered micrograph (A and B).

Fig. 3

Localization of NGFRs in the human foetal cornea. Human foetal (A–F) and adult (G and H) corneas were serially sectioned, incubated with anti-p75 (A, clone 7F10; C and D, clone NGFR5¹), anti-TrkA (B), anti-NGF (B, inset), anti-p63 (E and G), anti-ABCG2 (F and H) or anti-CK3/12 (F and H, insets) antibodies and processes for immunochistochemistry. Arrows in (A and B) identify morphologically distinct tissue architecture corresponding to the foetal limbus. Arrowheads in (C) point to immunoreactive limbal blood vessels, while those in (D) identify nerve fibres. Arrows in (E and F) point to occasional p63 reactive and faint ABCG2 stained foetal limbal cells. All tissue was counterstained identically, but immunoreactivity developed with diverse chromogens (A–D, DAB [brown]; E–H, AEC [red]). Results are representative of all adult and foetal specimens assessed. Original magnification ×400 (A and B), and ×1000 (C–H).

Fig. 4

Localization of NGFRs in cultured ocular surface epithelial cells. Human conjunctival (CEC-GB3, A and B), limbal (LEC-PC, C and D) and pterygium (PEC-PW, E and F) epithelial cells were subcultured, each collected at passage 5, fixed and processed in paraffin blocks. Sections from each block were incubated with anti-p75 (A, C and E, clone 7F10), anti-TrkA (B, D and F) or an isotype control antibody (D, inset). Immunoreactivity and counter-staining was identical to sections displayed in Figs. 1–3. Arrows (A, C, E) indicate intense p75 membrane-associated staining on a small percentage of cells. Original magnification ×1000.

Fig. 5

Flow cytometric analysis of SC markers and p75 in cultured limbal epithelial cells. Histogram plots were generated from at least 100,000 enzymatically dissociated human corneal-limbal cells from passage 3 (P3) and passage 4 (P4) that were labelled with mouse monoclonal (p75 [A and D], p63 [B and E], ABCG2 [C and F], red histograms) or an appropriate isotype control antibody (A–F, blue histograms).

Fig. 6

Localization of p75 in the normal human epidermis. Epidermal specimens (tissue 1, A–C; tissue 2, D; tissue 3, E) were sectioned and incubated with an anti-human p75 monoclonal antibody (clone 7F10, A–E). Intense epidermal p75 staining developed in specific clusters of cells located in shallow rete ridges (A–C, arrowheads) while membraneous reactivity diminished in basal cells of the deeper rete ridges (B and C; red arrows). Black arrows in (C) identify subcutaneous nerve fibres. Vertical sections (D) through the skin identified intact hair follicles where intense reactivity for p75 was noted in the bulge (* asterisks) and dermal papillae (dp) but diminished in regions closest to the epidermis (epi). In transverse sections (E), p75 was highly expressed in the outer root sheath, while reactivity diminished in cells closest to the hair follicle orifice. The boxed region in (b and c) is magnified in (B and C, respectively). The area encompassed in (inset e) is magnified in (E). Sections were stained and counterstained as in Figs. 1–4. Original magnification (×100, A and D; ×200, inset e; ×400, B and C; ×1000, E).