A. Elovic

Expression of transforming growth factors-α and β1 messenger RNA and product by eosinophils in nasal polyps

Abstract Nasal polyps are thought to develop as a manifestation of a chronic inflammatory process involving the upper airways. The eosinophil characteristically represents a prominent component of the inflammatory cell infiltrate of these lesions. However, the major clinical problem associated with nasal polyps, nasal obstruction, reflects the proliferation of the stromal and epithelial elements, which constitute the bulk of these lesions. We recently reported that blood eosinophils of patients with hypereosinophilia can produce the cytokines transforming growth factors-α (TGF-α) and β 1 (TGF-β 1 ). These cytokines have many biologic activities, which include the regulation of epithelial proliferation, the promotion of extracellular matrix formation, and the induction of angiogenesis. We therefore used in situ hybridization to determine whether the eosinophils that infiltrate nasal polyps express TGF-α and/or TGF-β 1 messenger RNA and used immunohistochemistry to determine whether these eosinophils also express TGF-α and TGF-β 1 , proteins. We found that eosinophils represented a major source of both transforming growth factors in each case of nasal polyposis examined and that in most cases the majority of all eosinophils expressed both TGF-α and TGF-β 1 . These results suggest that production of TGF-α and TGF-β 1 by the infiltrating eosinophils may contribute to some of the pathologic changes observed in nasal polyposis, such as thickening of the epithelial basement membrane, stromal fibrosis, angiogenesis, and epithelial and glandular hyperplasia.

Eosinophil ablation and tumor development

Tissue eosinophilia in squamous cell carcinoma has long been recognized; however, the role of eosinophils in tumor development remains unclear. Studies have reported both favorable and unfavorable prognoses for patients with tumors exhibiting tumor-associated tissue eosinophilia (TATE). This study seeks to elucidate the potential role of the eosinophil in squamous cell carcinoma development and provide an experimental model for future studies. The carcinogen-induced hamster oral cancer model was found to fulfill these objectives. Eosinophils progressively infiltrate into this carcinogen-induced oral cancer model. We now demonstrate that TATE is completely abolished by the use of an anti-interleukin-5 monoclonal antibody (mAb) preparation, TRFK-5. Clinical observations revealed that TRFK-5-treated hamsters exhibited smaller tumor burden and delayed onset of tumor development. The results suggest that anti-interleukin-5 antibody treatment may delay and/or inhibit tumor development, and that eosinophils may have a tumor-promoting role.

IL-1β and IL-6 in mouse parotid acinar cells: characterization of synthesis, storage, and release

Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1β (IL-1β) and the anti-inflammatory cytokine IL-6 have not been established in normal exocrine gland secretory cells. Parotid glands and isolated acinar cells prepared from BALB/c mice were homogenized for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). IL-1β and IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on supernatants prepared from mouse parotid acinar cell (MPAC) preparations unstimulated or stimulated between 0 and 10 min with 10-5 M norepinephrine at 37°C. MPACs were fixed in paraformaldehyde, frozen sectioned for light and electron microscopy, and labeled with antibodies to IL-1β and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6 (614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant increase in IL-1β ( P < 0.03) and IL-6 ( P < 0.01) release into supernatants by 10 min that paralleled the time course of amylase release. In situ hybridization showed the presence of transcripts for IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1β and IL-6 were significantly higher ( P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1β and IL-6 and release these cytokines from their granules after α- and β-adrenergic stimulation.

Lack of TGF-α and TGF-β1 synthesis by human eosinophils in chronic oral ulcers

We recently demonstrated that eosinophils infiltrate prominently into cutaneous wounds in the Syrian hamster and represent a source of transforming growth factor-alpha and transforming growth factor-beta. In this study, we assessed the role of the eosinophil and eosinophil-derived transforming growth factors in human oral ulcers that exhibit delayed healing, descriptively termed traumatic ulcerative granuloma with stromal eosinophilia. Our aim was to determine whether eosinophils, which characteristically infiltrate traumatic ulcerative granuloma with stromal eosinophilia lesions, produced transforming growth factor-alpha or transforming growth factor-beta. Twelve cases of traumatic ulcerative granuloma with stromal eosinophilia were examined for transforming growth factor-alpha and transforming growth factor-beta mRNA and cellular protein by in situ hybridization and immunohistochemistry. Eosinophils in 92% of the cases did not express detectable cellular levels of mRNA for either of the transforming growth factors. In addition, only a small percentage of the many eosinophils infiltrating these lesions produced transforming growth factor-alpha or transforming growth factor-beta. The lack of significant synthesis of transforming growth factors by eosinophils in most of the cases of traumatic ulcerative granuloma with stromal eosinophilia is in striking contrast to the expression of transforming growth factors by the eosinophils that infiltrate the animal wound-healing model. Our findings may help to explain the delayed healing that is typical of TUGSE lesions.

Prediction of human oral cancer radiation responsiveness by histone (H3) mRNA in situ hybridization: A preliminary report

PURPOSE Cell cycle kinetics are believed to be a key determinant in radiation responsiveness. However, histomorphologic analysis remains an unreliable method of identifying proliferating cells. In this study, the fraction of cells undergoing division within oral cancer biopsy samples was used to predict the responsiveness of the tumor to radiation therapy. PATIENTS AND METHODS Eighteen cases of T1 or T2 squamous cell carcinoma of the floor of the mouth with known clinical outcomes were identified. All were treated at the Massachusetts General Hospital with external beam radiation therapy alone. The fraction of proliferating cells was determined using in situ hybridization of histone (H3) mRNA expression. Tissue viability and mRNA status was verified using in situ hybridization for beta-actin mRNA expression. RESULTS Matching the fraction of oral tumor cells positively labeled for histone (H3) mRNA (histone labeling index or HLI) with the actual clinical outcome showed that the HLI of radioresponsive oral tumors (12 cases) was 0.336+/-0.185 (approximately 34%+/-19%), whereas that for radioresistant oral tumors (six cases) was 0.088+/-0.078 (approximately 9%+/-7.8%). Using t-test statistical analysis for unpaired samples showed that the difference in HLI between the two groups was significantly different (P=.0068). CONCLUSIONS It is concluded that the use of in situ detection of histone (H3) mRNA may be a useful adjunctive criterion in the choice of treatment for human oral cancer.