NF-kappaB in cultivated middle ear epithelium.

OBJECTIVE: Otitis media with effusion (OME) may develop into a chronic course in some patients. In the majority of cases lipopolysaccharide (LPS) is detected in the middle ear fluid being capable of initiating and maintaining the inflammatory process associated with the disease. In addition, various extracellular signs of the inflammatory cascade are present, such as cytokines and ICAM-1 receptors. However, the underlying regulatory mechanisms situated intracellularily are sparsely recognized. NF-kappaB is a ubiquitously transcription factor complexed to its inhibitor within the cytoplasm. In response to stimuli, e.g. LPS or cytokines, NF-kappaB becomes activated and is translocated to the nucleus. At this level it induces transcription of various genes and subsequent expression of mRNA encoding for many immunoglobulins and cytokines, e.g. interleukin 8 (IL-8) which may be responsible for prolonging and maintaining the inflammatory process. METHODS: To evaluate if NF-kappaB is present in middle ear epithelium including to identify possible stimulating factors in vitro studies using rabbit middle ear epithelial cells (meec) were undertaken. Both normal cells and LPS exposed cells were studied. ELISA techniques were applied to detect NF-kappaB, ICAM-1 receptors and IL-8. All measurements were adjusted to the concentration of total cell protein (TP). RESULTS: NF-kappaB was detected in the normal middle ear epithelium in concentrations between 16.8 and 28.6 ng/microg TP. In response to LPS the NF-kappaB content increased with 25-33%. This enhancement became more pronounced with longer duration of the LPS exposition. A relatively distinct expression of ICAM-1 preceded the NF-kappaB increase, after which the ICAM-1 measurements declined. IL-8 was hardly measurable in normal cells. The IL-8 concentrations were higher in LPS exposed cultures. The time related curve demonstrated a diphasic shape with an early and a late maximum. CONCLUSION: The chronic inflammation seen in some OME patients may be due to LPS activating ICAM-1 receptors and NF-kappaB followed by release of IL-8. An autocrine pathway may be established through activation of TNF alpha and IL-1beta. Thus, elucidation of alternative pathways in the inflammatory cascade and elimination of LPS, alternatively inhibition of NF-kappaB and/or IL-8, should be an issue for future investigation.

Nuclear factor-kappaB in middle ear epithelial cells: a methodological study using an ELISA.

OBJECTIVE: To contribute to the elucidation of the intracellular mechanisms relating to otitis media with effusion (OME) by detecting and quantifying nuclear factor (NF)-kappaB in middle ear epithelial cells by means of an ELISA. MATERIAL AND METHODS: Using an ELISA technique, NF-kappaB was identified and quantified in the cytoplasm of cultured epithelial cells from the rabbit middle ear exposed to a well-known inflammatory stimulus: lipopolysaccharide (LPS). RESULTS: NF-kappaB was measurable with the ELISA in a concentration range of 16-32 ng/microg total cell protein. LPS caused significant enhancement of NF-kappaB. Furthermore, the NF-kappaB concentration increased with the duration of LPS exposure. Western blot analysis confirmed the results obtained by ELISA. CONCLUSION: Further studies are required to determine the role of NF-kappaB in initiating and regulating the inflammatory response in the middle ear, including the significance of hyperoxia.