¹8O-Labeled nitrous acid and nitrite: Synthesis, characterization, and oxyhemoglobin-catalyzed oxidation to ¹8O-labeled nitrate.

We describe a simple laboratory method for specific labeling of nitrite with ¹8O for use in chemical and biochemical studies in the area of nitric oxide research. NaNO2 (0.1 mmol) is diluted in H2¹8O (45 µl) and acidified with HCl (1 µl, 5 M), and the solution is allowed to equilibrate. Subsequently, the sample is mixed by vortexing with ethyl acetate (500 µl), and the organic phase is dried over anhydrous Na2SO(4). Ethyl acetate is evaporated to dryness, and the residue is reconstituted in phosphate-buffered saline. In human blood hemolysate, oxyhemoglobin (HbFe¹6O2) was shown to oxidize N¹8O2⁻ to ¹6ON¹8O2⁻.

A disturbed interaction with accessory cells upon opportunistic infection with Pseudomonas aeruginosa contributes to an impaired IFN-γ production of NK cells in the lung during sepsis-induced immunosuppression.

Impaired resistance to Pseudomonas aeruginosa-induced pneumonia after cecal ligation and puncture (CLP), a mouse model for human polymicrobial sepsis, is associated with decreased IFN-γ, but increased IL-10, levels in the lung. We investigated the so far unknown mechanisms underlying this reduced IFN-γ synthesis in CLP mice. CD11b(+) NK cells, but not T or NKT cells in the lung were impaired in IFN-γ synthesis upon challenge with Pseudomonas in vitro and in vivo after CLP. The inhibition of NK cells was independent of IL-10. IFN-γ synthesis of NK cells was only partly restored by addition of recombinant IL-12. Accessory cells including dendritic cells and alveolar macrophages were required for maximal IFN-γ secretion. But accessory cells of CLP mice suppressed the IFN-γ secretion from naive lung leukocytes. In turn, naive accessory cells were unable to restore the IFN-γ production from lung leukocytes of CLP mice. Thus, a disturbed interaction of accessory cells and NK cells is involved in the impaired IFN-γ release in response to Pseudomonas in the lung of CLP mice. Considering the importance of IFN-γ in the immune defense against bacteria the dysfunction of accessory cells and NK cells might contribute to the enhanced susceptibility to Pseudomonas after CLP.