interleukin-8 and acetylleucyl-leucyl-norleucinal

Monocytic cells were stimulated with IgG-OVA equivalence immune complexes, mAb reacting with FcgammaRI, FcgammaRIIA, and FcgammaRIII, LPS, TNF-alpha, and the combination of ionomycin and phorbol ester, to address their effects on the expression of the mRNAs encoding for chemokines. Stimulation of monocytes with immune complexes induced a rapid expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and IL-8 mRNAs. In contrast, RANTES mRNA was already detectable in resting cells and only increased after 16 h of stimulation. A similar pattern was observed following homotypic stimulation of FcgammaR with mAb reacting with FcgammaRI and FcgammaRIIA, but not with a mAb reacting with FcgammaRIII, a subtype of receptor not expressed in THP-1 cells, thus indicating that both FcgammaRI and FcgammaRIIA are involved in the response. The pattern of chemokine induction elicited by LPS and the combination of ionomycin and PMA showed some similarities to those produced by FcgammaR cross-linking, although expression of IFN-gamma-inducible protein 10 mRNA was also observed in response to those agonists. The production of MIP-1alpha, MIP-1beta, and RANTES proteins encompassing the induction of their mRNAs was confirmed by specific ELISA. Experiments to address the transcription factors involved in the regulation of MIP-1alpha using pharmacological agents and EMSA showed the possible involvement of CCAAT/enhancer-binding protein beta sites and ruled out the functional significance of both NF-AT and AP-1 sites.

Topics: Antigen-Antibody Complex; Base Sequence; Chemokine CCL3; Chemokine CCL4; Chemokine CCL5; Cross-Linking Reagents; Gene Expression Regulation; Humans; Interleukin-8; Leupeptins; Macrophage Activation; Macrophage Inflammatory Proteins; Molecular Sequence Data; Monocytes; NF-kappa B; Receptors, IgG; RNA, Messenger; Salicylates; Tumor Cells, Cultured

The alveolar macrophage-derived peptide tumor necrosis factor-alpha (TNFalpha) initiates pulmonary inflammation through its ability to stimulate interleukin-8 (IL-8) synthesis in alveolar epithelial cells through an incompletely described transcriptional mechanism. In this study, we use the technique of ligation-mediated polymerase chain reaction (LMPCR) to record changes in transcription factor occupancy of the IL-8 promoter after TNFalpha stimulation of A549 human alveolar cells. Using dimethylsulfate/LMPCR, no detectable proteins bind the TATA box in unstimulated cells. By contrast, TNFalpha rapidly induces protection of G residues at -79 and -80 coincident with endogenous IL-8 gene transcription. Using DNase I/LMPCR, we observe inducible protection of nucleotides -60 to -99 (the TNF response element) and nucleotides -3 to -32 (containing the TATA box). Surprisingly, extensive TATA box protection is only seen after TNFalpha stimulation. Using a two-step microaffinity isolation/Western immunoblot DNA binding assay, we observe that the NF-kappaB subunits Rel A, NF-kappaB1, and c-Rel inducibly bind the TNF response element; these proteins undergo rapid TNFalpha-inducible increases in nuclear abundance as a consequence of IkappaBalpha proteolysis. Furthermore, the peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent that blocks both IkappaBalpha proteolysis and NF-kappaB subunit translocation, abrogates recombinant human TNFalpha-inducible IL-8 gene transcription. These studies demonstrate that IL-8 is activated by a promoter recruitment mechanism in alveolar epithelial cells, where NF-kappaB subunit translocation is required for (and coincident with) binding of the constitutively active TATA box-binding proteins.

Topics: Binding Sites; Blotting, Western; Cell Line; Epithelial Cells; Humans; Hydrolysis; Interleukin-8; Kinetics; Leupeptins; Lung; Promoter Regions, Genetic; Transcription Factors; Transcription, Genetic; Tumor Necrosis Factor-alpha

Multiple cell adhesion proteins are up-regulated in vascular endothelial cells in response to TNF alpha and other inflammatory cytokines. This increase in cell adhesion gene expression is thought to require the transcription factor NF-kappa B. Here, we show that peptide aldehyde inhibitors of the proteasome, a multicatalytic protease recently shown to be required for the activation of NF-kappa B, block TNF alpha induction of the leukocyte adhesion molecules E-selectin, VCAM-1, and ICAM-1. Striking functional consequences of this inhibition were observed in analyses of leukocyte-endothelial interactions under defined flow conditions. Lymphocyte attachment to TNF alpha-treated endothelial monolayers was totally blocked, while neutrophil attachment was partially reduced but transmigration was essentially prevented.

Topics: Base Sequence; Calpain; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Cysteine Endopeptidases; E-Selectin; Endothelium, Vascular; Gene Expression; Humans; In Vitro Techniques; Intercellular Adhesion Molecule-1; Interleukin-8; Leukocytes; Leupeptins; Molecular Sequence Data; Multienzyme Complexes; NF-kappa B; Oligodeoxyribonucleotides; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; RNA, Messenger; Time Factors; Transcription Factor RelB; Transcription Factors; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1