The gene for human granulocyte/macrophage colony-stimulating factor (GM-CSF) is expressed in a tissue-speciflic as well as an activation-dependent manner. The interaction of nuclear proteins with the promoter region of the GM-CSF gene that is likely to be responsible for this pattern of GM-CSF expression was investigated. We show that nuclear proteins interact with DNA fragments from the GM-CSF promoter in a cell-specific manner. A region spanning two cytokine-specific sequences, cytokine 1 (CK-1, 5' GAGATTC-CAC 3') and cytokine 2 (CK-2, 5' TCAGGTA 3') bound two nuclear proteins [nuclear factor (NF)-GMa and NF-GMb]
Nuclear factor of activated T cells (NFAT) was originally described as a T-cell-specific transcription factor athat supported the activation of cytokine gene expression and mediated the immunoregulatory effects of cyclosporin A (CsA). As we observed that activated endothelial cells also expressed NFAT, we tested the antiinflammatory properties of CsA in endothelial cells. Significantly, CsA completely suppressed the induction of NFAT in endothelial cells and inhibited the activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene regulatory elements that use NFAT by 60%. CsA similarly mediated a reduction of up to 65% in GM-CSF mRNA and protein expression in activated endothelial cells. CsA also suppressed E-selectin, but not vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells, even though the E-selectin promoter is activated by NF-kappa B rather than NFAT. Hence, induction of cell surface expression of this leukocyte adhesion molecule by tumor necrosis factor (TNF)-alpha was reduced by 40% in the presence of CsA, and this was reflected by a 29% decrease in neutrophil adhesion. The effects of CsA on endothelial cells were also detected at the chromatin structure level, as DNasel hypersensitive sites within both the GM-CSF enhancer and the E-selectin promoter were suppressed by CsA. This represents the first report of NFAT in endothelial cells and suggests mechanisms by which CsA could function as an antiinflammatory agent.
The human interleukin-3 receptor (IL-3R) is expressed on myeloid, lymphoid, and vascular endothelial cells, where it transduces IL-3- dependent signals leading to cell activation. Although IL-3R activation may play a role in hematopoiesis and immunity, its aberrant expression or excessive stimulation may contribute to pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the generation and characterization of a monoclonal antibody (MoAb), 7G3, which specifically binds to the IL-3R alpha-chain and completely abolishes its function. MoAb 7G3 immunoprecipitated and recognized in Western blots the IL-3R alpha-chain expressed by transfected cells and bound to primary cells expressing IL-3R alpha. MoAb 7G3 bound the IL-3R alpha-chain with a kd of 900 pmol/L and inhibited 125I-IL-3 binding to high- and low-affinity receptors in a dose-dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited 125I-7G3 binding to high- and low-affinity IL- 3Rs, indicating that MoAb 7G3 and IL-3 bind to common or adjacent sites. In keeping with the inhibition of IL-3 binding, MoAb 7G3 antagonized IL-3 biologic activities, namely stimulation of TF-1 cell proliferation, basophil histamine release, and IL-6 and IL-8 secretion from human endothelial cells. Two other anti-IL-3R alpha-chain MoAbs failed to inhibit IL-3 binding or function. Epitope mapping experiments using truncated IL-3R alpha-chain mutants and IL-3R alpha/GM-CSFR alpha chimeras revealed that 31 amino acids in the N-terminus of IL-3R alpha were required for MoAb 7G3 binding. MoAb 7G3 may be of clinical significance for antagonizing IL-3 in pathologic conditions such as some myeloid leukemias, follicular B-cell lymphoma, and allergy. Furthermore, these results implicate the N-terminal domain of IL-3R alpha in IL-3 binding. Since this domain is unique to the IL-3/GM- CSF/IL-5 receptor subfamily, it may represent a novel and common binding feature in these receptors.
The adhesion of monocytes to vascular surfaces is central to inflammation and atherogenesis; however, very little is known about regulatory factors that can prevent these processes. Here we report the inhibition of human monocyte adhesion to human endothelial layers and plastic by interleukin-4 (IL-4), a T-cell-derived glycoprotein with pleiotropic activities. The inhibitory effects of IL-4 were seen with basal and cytokine-stimulated monocyte adhesion, were apparent at low concentration, and were abolished by inactivating IL-4. No direct toxic effect of IL-4 on monocytes was detected. Inhibition of adhesion was accompanied by small increases in monocyte surface expression of the leukocyte-functional antigen group of adhesion structures, suggesting that absolute levels of expression may be less important than the functional status of such molecules in the regulation of monocyte adhesion. In addition, inhibition by IL-4 of cytokine-stimulated monocyte adhesion was not associated with changes in the surface expression of cytokine receptors. These results suggest a role for IL-4 in the regulation of monocyte adhesion, and may provide for a common mechanism for the inhibitory effects of IL-4 on monocyte function.
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