[Tumor necrosis factor-alpha and interferon-gamma modulate in vitro expression of nitric oxide synthase in human...

Interest in the physiological, pathological and therapeutic implications of nitric oxide (NO) have grown exponentially, with human nasal cavity and paranasal sinuses considered a dominant source of NO, indicating that this molecule possesses the diversity of biological effects in the regulation of airway clearance and nonspecific cellular immunity. We previously observed differences in NO synthase (NOS) isoform constitutively expressed in nasal epithelial cells (NECs) from allergic and normal subjects.

We extended the previous work to determine whether in vitro stimulation with proinflammatory cytokines influences levels of different NOS isoform expression.

Nasal epithelial cells were sampled from the inferior turbinate in a group of 16 healthy normal controls and 11 patients with perennial allergic rhinitis against house dust (HD) mite antigen. 1 x 10(5) cells were incubated in conditioned medium for 24 hours. Human recombinant interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), or both (cytomix) were added to a final concentration of 10 ng/ml. Cells were then fixed with 4% paraformaldehyde and processed for fluorescence immunocytochemistry. Immunoreactivity for 2 NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), was studied by laser scanning confocal microscopy and fluorescence intensity was assessed quantitatively.

We observed constitutive eNOS expression in epithelial cells of all subjects. Different treatments with cytokines did not affect eNOS expression. Cytokine treatment, however, significantly augmented iNOS expression in the control group. The average increase induced with IFN-gamma, TNF-alpha, and cytomix was 1.8, 2.33, and 2.31-fold. Nasal epithelial cells in the HD group showed elevated steady-state iNOS expression even in untreated. Cytokine treatment did not affect the degree of iNOS expression in this group.

These results confirm our previous findings that nasal epithelial cells in patients with allergic rhinitis produce higher levels of NO through the concomitant expression of different NOS isoforms. We also demonstrated that nasal epithelial cells have the potential to express iNOS protein spontaneously or upon stimulation with inflammatory cytokines, such as IFN-gamma and TNF-alpha. Because the high level of exhaled NO is considered a potential marker of allergic airway inflammation, preserving the iNOS gene from its unregulated induction may be important for maintenance of nasal homeostasis and may offer a tool for therapeutic intervention.