The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% ؎ 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P ؍ 0.004) and more variable level of activity (29% ؎ 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a newly assigned member of the Ig-immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. In this study, we hypothesized that PECAM-1 plays an essential in vivo role as a counterregulator of immediate hypersensitivity reactions. We found that PECAM-1 was highly expressed on the surface of immature bone marrow mast cells and at a lower density on mature peritoneal mast cells. Examination of skin biopsies from PECAM-1+/+ and PECAM-1−/− mice revealed that absence of PECAM-1 did not affect mast cell development or the capacity of mast cells to populate tissues. To examine whether the absence of PECAM-1 would influence immediate hypersensitivity reactions, PECAM-1+/+ and PECAM-1−/− mice were presensitized with anti-DNP mouse IgE and then challenged 20 h later with DNP-BSA or PBS. PECAM-1−/− mice exhibited elevated serum histamine concentrations after Ag stimulation compared with PECAM-1+/+ mice, indicating an increased severity of systemic IgE-mediated anaphylaxis. PECAM-1−/− mice have increased sensitivity to local cutaneous IgE-dependent anaphylaxis compared with PECAM-1+/+ mice, as assessed by greater tissue swelling of their ears and mast cell degranulation in situ. PECAM-1−/− bone marrow mast cells showed enhanced dense granule serotonin release after FcεRI cross-linking in vitro. These results suggest that PECAM-1 acts as a counterregulator in allergic disease susceptibility and severity and negatively modulates mast cell activation.
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