Fig. 2.
Fig. 2. FcγR expression on AMs is regulated by levels of GM-CSF in the lungs and expression of PU.1 in AMs, and levels are increased during pulmonary infection by adenovirus. / (A) FcγR expression on primary AMs (GM+/+, GM−/−, SPC-GM+/+/GM−/−) or cultured AMs (MH-S, mAMGFP+, mAMPU.1+) was quantified by FACS as described in “Materials and methods.” Shown are data for FcγRII/III-specific antibodies (shaded histogram) and isotype controls (open histogram). Differences in autofluorescence among cultured AM cell lines, due in part to the presence of the GFP marker in mAMGFP+ and mAMPU.1+, have been compensated. Results are representative of 4 separate determinations in AMs from mice analyzed individually. (B) The cultured AM cell line from GM−/− mice (mAMGFP+) failed to express mRNA for either activating (FcγRIA, FcγRIIIA) or inhibiting (FcγRIIB) FcRs, but expression was stimulated by PU.1 as shown in mAMPU.1+ cells. Total RNA was prepared from cultured AM cell lines (MH-S, mAMGFP+, mAMPU.1+) and subjected to RT-PCR analysis using gene-specific primers as described in “Materials and methods.” Shown are photographs of ethidium bromide–stained RT-PCR reaction products separated on 2% agarose gels. H20 PCR controls were negative (not shown). This experiment was repeated twice with identical results. (C) Primary AMs were recovered 36 hours after pulmonary adenoviral infection of GM+/+ or GM−/− mice, and levels of FcγR expression were assessed as above. Levels of FcγR on AMs are represented as the mean fluorescence intensity as determined using the FcγRII/III-specific antibody. Data represent means ± SEM; n = 4 mice per group; AMs from each mouse were analyzed individually. Differences in FcγR expression on AMs from infected and uninfected GM+/+ mice were significant (P < .001). FcγR expression was not detected (*) on GM−/− AMs in the absence or presence of adenovirus infection. AMs recovered from GM−/− mice and exposed ex vivo to IFN-γ for 24 hours showed a marked up-regulation of cell-surface FcγRII/III expression at levels significantly different from untreated mice (P < .001).

FcγR expression on AMs is regulated by levels of GM-CSF in the lungs and expression of PU.1 in AMs, and levels are increased during pulmonary infection by adenovirus.

(A) FcγR expression on primary AMs (GM+/+, GM−/−, SPC-GM+/+/GM−/−) or cultured AMs (MH-S, mAMGFP+⁠, mAMPU.1+⁠) was quantified by FACS as described in “Materials and methods.” Shown are data for FcγRII/III-specific antibodies (shaded histogram) and isotype controls (open histogram). Differences in autofluorescence among cultured AM cell lines, due in part to the presence of the GFP marker in mAMGFP+ and mAMPU.1+⁠, have been compensated. Results are representative of 4 separate determinations in AMs from mice analyzed individually. (B) The cultured AM cell line from GM−/− mice (mAMGFP+⁠) failed to express mRNA for either activating (FcγRIA, FcγRIIIA) or inhibiting (FcγRIIB) FcRs, but expression was stimulated by PU.1 as shown in mAMPU.1+ cells. Total RNA was prepared from cultured AM cell lines (MH-S, mAMGFP+⁠, mAMPU.1+⁠) and subjected to RT-PCR analysis using gene-specific primers as described in “Materials and methods.” Shown are photographs of ethidium bromide–stained RT-PCR reaction products separated on 2% agarose gels. H20 PCR controls were negative (not shown). This experiment was repeated twice with identical results. (C) Primary AMs were recovered 36 hours after pulmonary adenoviral infection of GM+/+ or GM−/− mice, and levels of FcγR expression were assessed as above. Levels of FcγR on AMs are represented as the mean fluorescence intensity as determined using the FcγRII/III-specific antibody. Data represent means ± SEM; n = 4 mice per group; AMs from each mouse were analyzed individually. Differences in FcγR expression on AMs from infected and uninfected GM+/+ mice were significant (P < .001). FcγR expression was not detected (*) on GM−/− AMs in the absence or presence of adenovirus infection. AMs recovered from GM−/− mice and exposed ex vivo to IFN-γ for 24 hours showed a marked up-regulation of cell-surface FcγRII/III expression at levels significantly different from untreated mice (P < .001).

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