C-C chemokine ligand 2 gene expression in nasal polyp fibroblasts: possible implication in the pathogenesis of nasal polyposis
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Published on: February 28, 2019
Chia-Tung Shun1, Sze-Kwan Lin, Chi-Yuan Hong
1Dept of Otolaryngology, National Taiwan University Hospital, 1 Chang-Teh Street, Taipei, Taiwan.
Objectives:
Recruitment of macrophages is essential to the pathogenesis of nasal polyps (NP), since this disease is inflammation-related. In this study, the effects of tumor necrosis factor alpha (TNF-alpha) on the expression of C-C chemokine ligand 2 (CCL2) in fibroblasts derived from nasal polyps (NPFs) were investigated. The roles of cyclooxygenase (COX) 2 and prostaglandins in the mediation of TNF-alpha-stimulated CCL2 gene expression were also investigated.
Methods:
Northern blot analysis was used to study the expression of CCL2 and c-Fos in cultured NPFs. An electrophoretic mobility shift assay was used to explore the interactions between activator protein 1 (AP- 1) and DNA. Immunohistochemistry was used to explore the in vivo expressions of COX-2, CCL2, and CD68 in NPs.
Results:
The Northern blot analysis showed that TNF-alpha stimulated the expression of CCL2 and COX-2 genes, and the synthesis of CCL2 messenger RNA was COX-2-dependent. A transient elevation of c-Fos and c-Jun messenger RNAs was induced by TNF-alpha, whereas COX-2 inhibitors NS-398 and meloxicam abolished the up-regulation of c-Fos. The electrophoretic mobility shift assay revealed that TNF-alpha triggered AP-1 and DNA binding and again, NS-398 and meloxicam inhibited this reaction via reducing c-Fos synthesis. Curcumin (AP-1 inhibitor) markedly suppressed the TNF-alpha-induced CCL2 expression. The immunohistochemical staining of NP surgical specimens also revealed an intimate alignment between CCL2-positive fibroblasts and CD-68-positive macrophages.
Conclusions:
These data suggest that NPFs may contribute to NP development by synthesizing CCL2 to promote macrophage recruitment. Furthermore, COX-2 facilitates CCL2 transcription in NPFs via a c-Fos and AP-1 signaling pathway.
Insights
Nasal polyp fibroblasts synthesize C-C chemokine ligand 2 (CCL2) to recruit macrophages, a key step in nasal polyp development. Tumor necrosis factor alpha (TNF-alpha) stimulates this via cyclooxygenase-2 (COX-2) and the AP-1 pathway.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Macrophage recruitment is crucial in nasal polyp (NP) pathogenesis.
- Nasal polyps are inflammatory in nature, necessitating investigation into cellular signaling pathways involved in their development.
Purpose of the Study:
- To investigate the effect of tumor necrosis factor alpha (TNF-alpha) on C-C chemokine ligand 2 (CCL2) expression in nasal polyp fibroblasts (NPFs).
- To elucidate the roles of cyclooxygenase (COX) 2 and prostaglandins in mediating TNF-alpha-stimulated CCL2 gene expression.
Main Methods:
- Northern blot analysis to assess gene and messenger RNA expression of CCL2, c-Fos, and c-Jun in NPFs.
- Electrophoretic mobility shift assay (EMSA) to examine activator protein 1 (AP-1) DNA binding.
- Immunohistochemistry to evaluate in vivo expression of COX-2, CCL2, and CD68 in NP tissues.
Main Results:
- TNF-alpha significantly upregulated CCL2 and COX-2 gene expression in NPFs, with CCL2 synthesis being COX-2 dependent.
- TNF-alpha induced transient expression of c-Fos and c-Jun; COX-2 inhibitors (NS-398, meloxicam) and an AP-1 inhibitor (curcumin) suppressed TNF-alpha-induced CCL2 expression.
- In vivo analysis showed co-localization of CCL2-positive fibroblasts and CD68-positive macrophages in NP tissues, indicating a role in macrophage recruitment.
Conclusions:
- NPFs contribute to NP development by producing CCL2, which recruits macrophages.
- COX-2 plays a critical role in facilitating CCL2 transcription in NPFs through a signaling pathway involving c-Fos and AP-1.
