The functional characterization of lung-associated natural killer activity and its regulation by alveolar macrophages.

Abstract

The purpose of this study was to characterize the natural killer activity of lymphocytes resident in the rat lung and explore its modulation. Lung lymphocytes (LL) were shown to possess potent NK activity against Yac-1 targets, The effector cells were found to be sensitive to complement-mediated lysis with anti-asialo GM-1 and to treatment with L-leucine methyl ester, which selectively kill NK cells. LL were more potent NK effectors than peripheral blood (PBL) or spleen lymphocytes (SL), although the lung and spleen contained similar proportions of NK (3.2.3 positive) cells. Lung and peripheral blood NK activities were modulated to a similar extent by human recombinant interleukin-2 (IL-2) whereas spleen NK activity was significantly more responsive. Interferon-$\alpha\beta$ (IFN-$\alpha\beta$) increased NK activity in all three compartments but to a lesser extent in PBL. By contrast, interferon-$\gamma$ (IFN-$\gamma$) failed to augment lung or spleen NK activity but did significantly enhance NK activity in PBL. Interestingly, the regulatory effects of alveolar macrophages (AM) also varied depending upon the origin of the lymphocytes. A more thorough examination of the responsiveness of lung NK activity to cytokine treatment was undertaken once it had been established that LL behave differently than PBL or SL. Lung lymphocytes were treated in vitro for 18h with various cytokines. Human recombinant IL-2, purified rat IFN-$\alpha\beta$, or murine recombinant tumor necrosis factor-$\alpha$ (TNF-$\alpha$) resulted in a dose-dependent increase in lung NK activity. The maximum stimulation by IL-2 and IFN-$\alpha\beta$ was similar (${\simeq}55$%) though much lower concentrations of IL-2 were required for the same effect. TNF-$\alpha$ stimulation was approximately half that seen for IL-2 and was only observed at a high concentration of the cytokine. Furthermore, doses of IFN-$\alpha\beta$ and TNF-$\alpha$ that had little effect alone were able to synergize with suboptimal doses of IL-2. By contrast, lung NK activity was resistant to the influence of interleukin-1 (IL-1) and IFN-$\gamma$, alone or in combination with suboptimal doses of IL-2. The interaction of lung lymphocytes with alveolar macrophages may have a profound regulatory effect on NK activity. This interaction was explored using a two-chambered system which prohibited physical contact between the cells in the separate chambers but allowed diffusion of soluble factors. AM were found to inhibit LL NK activity in a time-dependent and reversible manner. AM inhibition was shown to be sensitive to indomethacin which caused a decrease in prostaglandin E$\sb2$ (PGE$\sb2$) concentration. Quantitation of PGE$\sb2$ levels and treatment with exogenous PGE$\sb2$ indicated that it could not account for the entire inhibitory effect. It was subsequently found that exogenous transforming growth factor-$\beta\sb1$ (TGF-$\beta\sb1$) also inhibits lung NK activity and that treatment of inhibitory AM supernatant with a neutralizing antibody to TGF-$\beta\sb1$ absorbs up to 55% of the inhibitory activity. Moreover, the co-culture supernatant was found to contain 24 pg/mL TGF-$\beta\sb1$, enough to account for the remainder of the inhibition. By contrast, PDGF and nitric oxide were shown not to be involved in the inhibition. The mechanism by which TGF-$\beta\sb1$ and thereby AM inhibit lung NK activity was investigated. (Abstract shortened by UMI.)