Abstract
Introduction
Nasal polyps (NP) are regulated by proinflammatory transcription factors such as activator protein-1 (AP-1), which comprises members of the proto-oncogene Jun and Fos protein families. The binding of AP-1 proteins to the 12-O-tetradecanoylphorbol-13-acetate (TPA)-response element can activate target genes and regulate many critical cellular processes. The proliferating cell nuclear antigen (PCNA) gene contains AP-1 sites, and its expression is regulated by AP-1 activity. In this study, NP and inferior turbinate (IT) were evaluated, compared with normal mucosa, to see if diffuse inflammation and active cellular proliferation exist.
Materials and Methods
A diseased group of 20 subjects and control group of 20 subjects were enrolled in this study. NP and IT were evaluated with expression of phospho-c-Jun, c-Fos, PCNA, major basic protein by immunohistochemistry, and eosinophil numbers by cell counts.
Result
The expression of phospho-c-Jun, c-Fos, PCNA, major basic protein, and eosinophil numbers showed no significant difference in IT and NP of the same patients, but all were significantly higher in IT and NP compared with normal mucosa ( P < .05).
Conclusion
Our result demonstrated strong evidence that diffuse mucosal inflammation and active cellular proliferation do exist in rhinosinusitis with nasal polyposis. As the degree of the disease severity increases, the difference of eosinophilic infiltration and cellular proliferation activity between NP and its adjacent mucosa decreases. An integrated anti-inflammatory treatment may be more important than surgical intervention.
1
Introduction
Nasal polyps (NP) are known to express high levels of multiple inflammatory genes regulated by proinflammatory transcription factors such as activator protein-1 (AP-1) . AP-1 is a hetero- or homodimeric complex that comprises members of the proto-oncogene Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) protein families. After a broad range of stimuli, AP-1 proteins move from the cytoplasm into the nucleus and bind to the 12-O-tetradecanoylphorbol-13-acetate (TPA)-response element to activate target genes. It can regulate many critical cellular processes, including cell proliferation, differentiation, apoptosis, and oncogenic transformation . The proliferating cell nuclear antigen (PCNA) is a marker for the G1-S transition in the cell cycle. The PCNA-labeled nuclei identify a population of cells in the phase of active proliferation . The PCNA gene contains AP-1 sites in the promoter region, and its expression is regulated by AP-1 activity . In a study by Lavezzi et al , PCNA overexpression in NP was investigated and was found to be significantly higher than that in normal sinus mucosa. Hence, it is reasonable to speculate that the expression of c-Jun and c-Fos (the most abundant AP-1 heterodimer) would increase and subsequently result in PCNA overexpression and active cellular proliferation in NP.
In the previous study, the pattern of inflammatory cell infiltrations in NP and the paired middle turbinate (MT) mucosa is similar, suggesting a diffuse pattern of mucosal inflammation . However, as we know, the MT is considered to be the common site of NP origin, and it is not surprising. If the diffuse mucosal inflammation or cell proliferation extends to other sinonasal mucosa, which is far from the origin of NP, it will provide a strong evidence for integrated anti-inflammatory therapy, not just functional surgery, in patients with NP. Therefore, to address this issue, we have used immunohistochemical techniques to assess mucosal samples from healthy sinus mucosa, NP, and its adjacent inferior turbinate (IT) tissues from Asian patients with bilateral chronic rhinosinusitis (CRS) and NP.
2
Materials and methods
2.1
Subjects
This study was approved by the ethics committee of Chang Gung Memorial Hospital, Taiwan (reference number, 95-1205B). In the disease group, 20 patients (13 men and 7 women) with a diagnosis of CRS with bilateral NP scheduled for surgical treatment were enrolled in this study. The mean age of the disease group was 45.3 years (ranging from 21 to 68 years). All patients had a preoperative computed tomographic scan with a score higher than 12 (according to the modified Lund-Mackay staging system ). The NP and IT tissues of the same patient were harvested for investigation. The control group consisted of 20 patients, 9 men and 11 women, aged from 29 to 63 years (mean age, 38.6 years), who received transsphenoidal resection for pituitary tumors without obvious sinus inflammation on magnetic resonance imaging scans. All the patients did not receive any intranasal or oral steroids or antibiotics therapy at least 1 month before the surgery and had no history of allergic rhinitis, asthma, cystic fibrosis, or aspirin intolerance. The ImmunoCAP system (Pharmacia, Uppsala, Sweden) was used to detect the atopic status.
2.2
Immunohistochemistry
Formalin- or paraffin-embedded tissues were sectioned by the department of pathology of Chang Gung Memorial Hospital. Four- μ m thickness slides were used for immunohistochemistry. All formalin-embedded sections were deparaffinized, rehydrated through serial alcohol, and antigen retrieval was performed. The slides were incubated with appropriate dilution of antibodies: major basic protein (MBP) (Chemicon Inc, Temecula, CA) for eosinophil, PCNA (NeoMarkers Inc, CA), c-fos (Santa Cruz Biotechnology Inc, Santa Cruz, CA), and phospho-c-jun (Santa Cruz) at room temperature for 1 hour. After incubation, these slides were washed with phosphate buffer solution for 3 times, incubated with horseradish peroxidase polymer antibody (Zymed Laboratories Inc, CA) at room temperature for 10 minutes, and developed by addition of diaminobenzidine at room temperature for 10 minutes.
2.3
Cell counting and staining score
Stained tissue sections were examined at ×400 magnification using an Olympus microscope (Olympus America Inc, CA) and photographed using the Arc digital software package (MIS Inc, Chicago, IL). Four fields with high-intensity positive-cell area (epithelium and submucosal glands) were selected in each section. The number of eosinophil per high-power field of magnification (×400) was calculated and averaged. The PCNA, c-fos, and phospho-c-jun staining were graded on a 4-point scale by randomly selecting 5 high-powered fields: point 1, 0% to 25%; point 2, 25% to 50%; point 3, 50% to 75%; point 4, 75% to 100%.
2.4
Statistical analysis
All statistical analyses were performed with the SPSS 12.0 statistical software program (IBM Corporation, Chicago, IL). The Wilcoxon signed rank test was used to compare paired samples from NP and IT of the same patients for statistical differences. The Mann-Whitney U test was used for comparing results from independent groups. Spearman’s rank correlation test was used to correlate the results of phospho-c-Jun, c-Fos, and PCNA expression and the results of eosinophils in NP. P < .05 was considered to be statistically significant.
2
Materials and methods
2.1
Subjects
This study was approved by the ethics committee of Chang Gung Memorial Hospital, Taiwan (reference number, 95-1205B). In the disease group, 20 patients (13 men and 7 women) with a diagnosis of CRS with bilateral NP scheduled for surgical treatment were enrolled in this study. The mean age of the disease group was 45.3 years (ranging from 21 to 68 years). All patients had a preoperative computed tomographic scan with a score higher than 12 (according to the modified Lund-Mackay staging system ). The NP and IT tissues of the same patient were harvested for investigation. The control group consisted of 20 patients, 9 men and 11 women, aged from 29 to 63 years (mean age, 38.6 years), who received transsphenoidal resection for pituitary tumors without obvious sinus inflammation on magnetic resonance imaging scans. All the patients did not receive any intranasal or oral steroids or antibiotics therapy at least 1 month before the surgery and had no history of allergic rhinitis, asthma, cystic fibrosis, or aspirin intolerance. The ImmunoCAP system (Pharmacia, Uppsala, Sweden) was used to detect the atopic status.
2.2
Immunohistochemistry
Formalin- or paraffin-embedded tissues were sectioned by the department of pathology of Chang Gung Memorial Hospital. Four- μ m thickness slides were used for immunohistochemistry. All formalin-embedded sections were deparaffinized, rehydrated through serial alcohol, and antigen retrieval was performed. The slides were incubated with appropriate dilution of antibodies: major basic protein (MBP) (Chemicon Inc, Temecula, CA) for eosinophil, PCNA (NeoMarkers Inc, CA), c-fos (Santa Cruz Biotechnology Inc, Santa Cruz, CA), and phospho-c-jun (Santa Cruz) at room temperature for 1 hour. After incubation, these slides were washed with phosphate buffer solution for 3 times, incubated with horseradish peroxidase polymer antibody (Zymed Laboratories Inc, CA) at room temperature for 10 minutes, and developed by addition of diaminobenzidine at room temperature for 10 minutes.
2.3
Cell counting and staining score
Stained tissue sections were examined at ×400 magnification using an Olympus microscope (Olympus America Inc, CA) and photographed using the Arc digital software package (MIS Inc, Chicago, IL). Four fields with high-intensity positive-cell area (epithelium and submucosal glands) were selected in each section. The number of eosinophil per high-power field of magnification (×400) was calculated and averaged. The PCNA, c-fos, and phospho-c-jun staining were graded on a 4-point scale by randomly selecting 5 high-powered fields: point 1, 0% to 25%; point 2, 25% to 50%; point 3, 50% to 75%; point 4, 75% to 100%.
2.4
Statistical analysis
All statistical analyses were performed with the SPSS 12.0 statistical software program (IBM Corporation, Chicago, IL). The Wilcoxon signed rank test was used to compare paired samples from NP and IT of the same patients for statistical differences. The Mann-Whitney U test was used for comparing results from independent groups. Spearman’s rank correlation test was used to correlate the results of phospho-c-Jun, c-Fos, and PCNA expression and the results of eosinophils in NP. P < .05 was considered to be statistically significant.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
