Evidence for distinct histologic profile of nasal polyps with and without eosinophilia

Abstract

Objective/hypothesis: To evaluate the histology, RNA, and protein signatures of nasal polyps (NPs) in order to demonstrate specific subtypes of disease and differentiate "idiopathic" NPs based on tissue eosinophilia.

Study design: Prospective laboratory-based study.

Methods: NP tissue was obtained from patients referred to the University of Virginia Health System for sinus surgery. Histology analyses included hematoxylin-eosin, Gomori's trichrome, toluidine blue, and chloroacetate staining. RNA and protein were extracted from tissue and cytokine transcript or protein concentrations determined.

Results: Idiopathic NPs can be divided into distinct subsets characterized by absence (NE) and presence (E) of prominent eosinophilia. The validity of this distinction is supported by the demonstration that NE polyps are further distinguished by glandular hypertrophy, dense collagen deposition, and mononuclear cellular infiltrate. In contrast, E-NP display edema, rare glandularity, and minimal collagen deposition except within the basement membrane. Total mast cell numbers were reduced in E-NP, whereas connective tissue mast cells were increased in NE-NP. Consistent with the distinctive pattern of increased fibrosis, NE-NP displayed increased transforming growth factor-β and vascular endothelial growth factor transcripts. Similarly, NE-NPs had higher concentrations of transforming growth factor-β, fibroblast growth factor-β, and platelet-derived growth factor protein.

Conclusions: Idiopathic NPs can be distinguished by NE and E and are supported by the observations that these display distinct histologic, gene, and protein expression patterns. The findings suggest that as unique diseases, idiopathic NPs will require distinct therapeutic interventions.

Figure 1

Eosinophil scoring of nasal polyp tissue. Tissue sections were examined at 400× magnification and the average number of eosinophils per high-powered field were counted in a blinded fashion. *p<0.001 as compared to control; **p<0.05 as compared to E-NP or AFS.

Figure 2

Determination of extracellular matrix content of nasal polyp tissue. Extracellular matrix/collagen content was scored using morphometric analysis with NIH Image J software. Color digital pictures were converted to 8-bit grey scale. Threshold values were set to exclude air and nuclei, with the remaining grey corresponding to extracellular matrix/collagen. Data are presented as mean grey volume (MGV). *p<0.01 as compared to control

Figure 3

Quantification of mast cell numbers in nasal polyp tissue. Tissue samples were fixed in 4% paraformaldehyde, sectioned and stained with toluidine blue (white bars) or chloroacetate esterase (CAE; black bars). Tissue sections were examined at 400× magnification and the average number of mast cells per high-powered field were counted in a blinded fashion. *p<0.01 as compared to control; **p<0.03 as compared to control

Figure 4

Quantitative assessment of transcript levels in nasal polyp tissue. Tissue samples were homogenized and RNA extracted. Transcript levels were quantified using PCR with sybr-green detection. Data were analyzed as the change in C_T of each cytokine transcript to ß-actin allowing comparison of control tissue and polyp tissue; TGF-ß2 black bars, VEGF grey bars and HIF-1α white bars. *p<0.05 as compared to NE; **p<0.03 as compared to NE

Figure 5

Comparison of fibrotic growth factors in nasal polyp tissue. Tissue samples were homogenized and cleared by centrifugation to remove debris. Cytokine concentrations were determined using either an enzyme-linked immunosorbent assay or cytometric bead-suspension assay and individual cytokine measurements were normalized to total protein to allow comparison between samples. Data is presented as pg/mg of total protein: VEGF hatched bars, FGF grey bars, PDGF black bars and TGF-ß2 white bars. *p<0.05 as compared to NE; p<0.002 as compared to NE