We have previously reported that MH-S, an established murine alveolar macrophage-derived cell line, mediated profound inhibition of in vitro antibody production, as did their freshly isolated alveolar macrophage (AM) counterparts. In this communication we show that like freshly recovered AMs, the MH-S cell line also displays phenotypic and functional heterogeneity. Sorting of parental MH-S cells by flow cytometry based on reactivity with anti-Mac-1 antibody yielded two subsets. Further analysis by staining with monoclonal antibodies against well-characterized murine macrophage cell surface markers revealed that both Mac-1+ and Mac-1- subsets expressed the mature murine macrophage antigen (F4/80) and class II major histocompatibility complex molecules, but with different intensity. In contrast, the two subsets stained equivalently with antibody against the Fc gamma II receptor, whereas neither subset stained with anti-CD4 antibody. Examination by light microscopy revealed plemorphism in the Mac-1+ population with many of the cells appearing spindle shaped and having elongated processes, whereas a majority of the cells in the Mac-1- population were spherical in shape. Functionally, cells from the Mac-1+ population were less inhibitory of in vitro antibody production and produced significantly more nitric oxide in response to stimulation with lipopolysaccharide than were cells in the Mac-1- population. Essentially similar results were obtained using cloned Mac-1+ and Mac-1- MH-S cells. The finding of heterogeneity in an established cell line that displays functions similar to those of freshly recovered AMs suggests that distinct subsets of AMs may be involved in the pathogenesis of disease processes in the lung.
Peritoneal and splenic adherent macrophages (SAC) from M. lepraemurium susceptible (C3H/HeJ) and resistant (C57B1/6J) mice were studied for their abilities to generate H2O2 in vitro. Unexpectedly, SAC from the susceptible C3H/HeJ strain produced more H2O2 than those of the resistant C57BL/6J. In vivo sensitization with M. bovis (BCG), or C. parvum increased production of H2O2 by SAC from both strains, whereas in vivo sensitization with M. lepraemurium enhanced H2O2 production only in the C3H/HeJ susceptible strains. In vitro addition of a crude lymphokine enhanced H2O2 production by C3H/HeJ SAC more than by C57BL/6J SAC. In vitro addition of M. lepraemurium caused an inhibition of H2O2 production by SAC from both strains but the inhibition was greater for the resistant C57BL/6J strain. M. lepraemurium phagocytosed in vitro by untreated peritoneal macrophages of both mouse strains were morphologically altered to the same extent. However, the addition of lymphokine dramatically increased the degree of bacterial lysis in only the C57BL/6J strain. These results, support the view that H2O2 plays a limited, if any, role in the protection of the host from M. lepraemurium and may even contribute to susceptibility by inhibiting the host's immune response.
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