We previously demonstrated using noninvasive technologies that the interferon-gamma (IFN-γ) receptor complex is preassembled [1]. In this report we determined how the receptor complex is preassembled and how the ligand-mediated conformational changes occur. The interaction of Stat1 with IFN-γR1 results in a conformational change localized to IFN-γR1. Jak1 but not Jak2 is required for the two chains of the IFN-γ receptor complex (IFN-γR1 and IFN-γR2) to interact; however, the presence of both Jak1 and Jak2 is required to see any ligand-dependant conformational change. Two IFN-γR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-γR2 with IFN-γR1. These results agree with a detailed model of the IFN-γ receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.

The contribution of interstitial macrophages (IM) to lung defense, homeostasis, and pathophysiology is for the most part unknown. Studies on this cell type are difficult because they are not readily accessible in large numbers or in high purity. In the present work, various nonenzymatic and enzymatic methods were compared with the aim of isolating and characterizing pure populations of lung IM. The results of our studies demonstrate that most procedures currently used to isolate IM yield subpopulations of alveolar macrophages (AM) or IM highly contaminated by AM and granulocytes. We found that lavage of the lung yielded only one half of the total AM present in the tissue. The remainder of the AM could only be obtained by extensive washing of cut and disaggregated lung tissue, which is considered by some investigators to be an effective procedure for IM isolation. According to our results, cells recovered by lavage and washing of cut and disaggregated lung tissue were morphologically and histochemically identical, were strongly positive for nonspecific esterase, highly phagocytic, and appeared to represent subpopulations of AM with apparently varying degrees of adherence to the alveolar walls. We also found that IM could be obtained in high purity by sequential digestion of the remaining lung tissue with 60 and 175 IU/ml of collagenase followed by selective adherence. Digestion of the tissue with 60 IU/ml of collagenase resulted in a highly enriched population of granulocytes and also reduced their contamination in the IM population. The resulting IM were distinct from AM by morphology and histochemistry. Like AM, these cells displayed Fc receptor-mediated phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The specific function of interstitial macrophages (IM) in the lung is poorly understood because of difficulties in isolating these cells in high purity or large number. In the present studies, a pure population of enzymatically isolated IM and lung macrophages obtained mechanically from the lung were compared functionally with alveolar macrophages recovered by lavage (AM). Macrophages isolated mechanically from the tissue and AM displayed similarly high levels of Fc-receptor mediated phagocytosis. In contrast, IM phagocytized significantly fewer opsonized sheep red blood cells per macrophage than AM. In addition, although some variations in the amounts of nitric oxide and superoxide anion produced by AM and macrophages obtained by mechanical tissue disruption were observed, these subpopulations released significantly more of these mediators than IM. These data support the concept that macrophages isolated by mechanical disruption of the tissue represent a subpopulation of AM. We also found that, in contrast to AM, IM did not respond synergistically to combinations of IFN-gamma and lipopolysaccharide (LPS) or tumor necrosis factor alpha in terms of nitric oxide production. Furthermore, regulation of superoxide anion release in AM and IM by LPS and/or IFN-gamma was distinct. Taken together, these studies demonstrate that IM are functionally different from other macrophage subpopulations which might reflect their unique location within the lung.

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The pathways regulating rat and mouse embryonic and lung fibroblast nitric oxide production were analyzed in an attempt to evaluate the potential role of these cells in nonspecific host defense and inflammation. Interleukin-1 beta (IL-1 beta) was found to be the strongest single activator in all types of fibroblasts examined. In addition, lipopolysaccharide (LPS) was synergistic with IL-1 beta or tumor necrosis factor-alpha (TNF-alpha) in induction of nitric oxide synthesis. These patterns of responsiveness are not observed in macrophages and may be significant in initiation of early host defense processes, before specific interferon-gamma (IFN-gamma)-mediated immune responses have become operative. Rat and mouse fibroblasts were also found to produce nitric oxide when primed with IFN-gamma and simultaneously treated with IL-1, TNF-alpha, or LPS. The doses of IFN-gamma effective in priming fibroblasts for nitric oxide production were as low as 1-10 U/ml. Furthermore, effective triggering doses of LPS, TNF-alpha, and IL-1 were 10 ng/ml, 100 U/ml, and 0.2 ng/ml, respectively. These results demonstrate that fibroblasts are activated more readily to produce nitric oxide than interstitial macrophages and may be the major source of this mediator in tissues. Immunohistochemical studies demonstrated that fibroblasts are heterogeneous with respect to inducible nitric oxide synthase expression with the majority of cells not involved in the response. Fibroblasts were also found to be distinct from macrophages in their sensitivity to the suppressive effects of transforming growth factor-beta, which in fibroblasts inhibited both IFN-gamma plus LPS- and IFN-gamma plus TNF-alpha-induced nitric oxide production. At the stage of growth crisis, a dramatic increase in nitric oxide production was observed in rat fibroblasts in response to IFN-gamma or TNF-alpha that may be directly correlated with cellular senescence. Taken together, our data suggest that mouse and rat fibroblasts are potential effectors in both IFN-gamma-dependent and -independent nitric oxide-mediated processes and that the patterns regulating nitric oxide metabolism in these cells are distinct from those of macrophages.