Ivan J. D. Lindley

Expression of monocyte chemotactic protein and interleukin-8 by cytokine-activated human vascular smooth muscle cells.

The present study was designed to investigate the capacity of human vascular smooth muscle cells (SMCs) to produce a cytokine chemotactic for monocytes (monocyte chemotactic protein [MCP]) and by way of comparison, a related polypeptide activator of neutrophils (known as interleukin-8 [IL-8] or neutrophil activating protein-1 [NAP-1]. On exposure to IL-1, SMCs released high levels of chemotactic activity for monocytes, which could be removed by absorption with anti-MCP antibodies. MCP production by activated SMCs was comparable to that of IL-1-stimulated umbilical vein endothelial cells. Activated SMCs released appreciable levels of IL-8, as determined by a specific enzyme-linked immunosorbent assay, but little chemotactic activity for neutrophils. IL-1-treated SMCs expressed high levels of both MCP and IL-8 mRNA transcripts, as assessed by Northern blot analysis. Tumor necrosis factor and bacterial lipopolysaccharide but not IL-6 also induced MCP and IL-8 gene expression in SMCs. Nuclear runoff analysis revealed that IL-1 augmented transcription of the MCP and IL-8 genes. The capacity of SMCs to produce a cytokine (MCP) that recruits and activates circulating mononuclear phagocytes may be of considerable importance in the pathogenesis of vascular diseases (e.g., vasculitis and atherosclerosis) that are characterized by monocyte infiltration of the vessel wall.

Neutrophils are indispensable for hematopoietic stem cell mobilization induced by interleukin-8 in mice

The CXC chemokine interleukin-8 (IL-8/CXCL8) induces rapid mobilization of hematopoietic progenitor cells (HPCs). Previously we showed that mobilization could be prevented completely in mice by pretreatment with neutralizing antibodies against the β2-integrin LFA-1 (CD11a). In addition, murine HPCs do not express LFA-1, indicating that mobilization requires a population of accessory cells. Here we show that polymorphonuclear cells (PMNs) serve as key regulators in IL-8-induced HPC mobilization. The role of PMNs was studied in mice rendered neutropenic by administration of a single injection of antineutrophil antibodies. Absolute neutropenia was observed up to 3–5 days with a rebound neutrophilia at day 7. The IL-8-induced mobilizing capacity was reduced significantly during the neutropenic phase, reappeared with recurrence of the PMNs, and was increased proportionally during the neutrophilic phase. In neutropenic mice, the IL-8-induced mobilizing capacity was restored by the infusion of purified PMNs but not by infusion of mononuclear cells. Circulating metalloproteinase gelatinase B (MMP-9) levels were detectable only in neutropenic animals treated with PMNs in combination with IL-8, showing that in vivo activated PMNs are required for the restoration of mobilization. However, IL-8-induced mobilization was not affected in MMP-9-deficient mice, indicating that MMP-9 is not indispensable for mobilization. These data demonstrate that IL-8-induced mobilization of HPCs requires the in vivo activation of circulating PMNs.

Chemokines: new ligands, receptors and activities

Abstract Chemokines, a large and growing family of structurally related proteins, were recognized originally for their ability to dictate the migration and activation of selected leukocyte populations. They now appear to have a number of additional roles, including T-cell development and activation, and angiogenesis. Current research interests in the area of chemokine biology were discussed at a recent meeting *

Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1

Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61β. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery.

1α,25-Dihydroxyvitamin D3 decreases DNA binding of nuclear factor-κB in human fibroblasts

1α,25‐Dihydroxyvitamin D3 (1,25‐(OH)2‐D3), the active metabolite of vitamin D, can inhibit NF‐κB activity in human MRC‐5 fibroblasts, targeting DNA binding of NF‐κB but not translocation of its subunits p50 and p65. The partial inhibition of NF‐κB DNA binding by 1,25‐(OH)2‐D3 is dependent on de novo protein synthesis, suggesting that 1,25‐(OH)2‐D3 may regulate expression of cellular factors which contribute to reduced DNA binding of NF‐κB. Although NF‐κB binding is decreased by 1,25‐(OH)2‐D3 in MRC‐5 cells, IL‐8 and IL‐6 mRNA levels are only moderately downregulated, demonstrating that inhibition of NF‐κB DNA binding alone is not sufficient for optimal downregulation of these genes.

Human macrophage inflammatory protein-3α/CCL20/LARC/Exodus/SCYA20 is transcriptionally upregulated by tumor necrosis factor-α via a non-standard NF-κB site

The 5′‐flanking sequences of the human macrophage inflammatory protein‐3α/CCL20 gene were cloned and transfected into G‐361 human melanoma cells in a luciferase reporter construct. Tumor necrosis factor‐α (TNF‐α) treatment stimulated luciferase expression, and promoter truncations demonstrated that TNF‐α inducibility is conferred by a region between nt −111 and −77, which contains a non‐standard nuclear factor‐κB (NF‐κB) binding site. The requirement for NF‐κB was demonstrated as follows: (i) mutations in this NF‐κB site abrogated TNF‐α responsiveness; (ii) TNF‐α activated a construct containing two copies of the CCL20 NF‐κB binding site; (iii) overexpression of NF‐κB p65 activated the CCL20 promoter; (iv) NF‐κB from nuclear extracts of TNF‐α‐stimulated cells bound specifically to this NF‐κB site.

Serpina1 is a potent inhibitor of IL-8-induced hematopoietic stem cell mobilization

Here, we report that cytokine-induced (granulocyte colony-stimulating factor and IL-8) hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is completely inhibited after low-dose (0.5 Gy) total-body irradiation (TBI). Because neutrophil granular proteases are regulatory mediators in cytokine-induced HSC/HPC mobilization, we considered a possible role for protease inhibitors in the induction of HSC/HPC mobilization. Bone marrow (BM) extracellular extracts that were obtained from murine femurs after 0.5 Gy of TBI contained an inhibitor of elastase. Also, after low-dose TBI, both Serpina1 mRNA and protein concentrations were increased in BM extracts, compared with extracts that were obtained from controls. The inhibitory activity in BM extracts of irradiated mice was reversed by addition of an Ab directed against Serpina1. To further study a possible in vivo role of Serpina1 in HSC/HPC mobilization, we administered Serpina1 before IL-8 injection. This administration resulted in an almost complete inhibition of HSC/HPC mobilization, whereas heat-inactivated Serpina1 had no effect. These results indicate that low-dose TBI inhibits cytokine-induced HSC/HPC mobilization and induces Serpina1 in the BM. Because exogenous administration of Serpina1 inhibits mobilization, we propose that radiation-induced Serpina1 is responsible for the inhibition of HSC/HPC mobilization. Also, we hypothesize that cytokine-induced HSC/HPC mobilization is determined by a critical balance between serine proteases and serine protease inhibitors.

Anti-IL-8 autoantibodies and complexes in rheumatoid arthritis: polyclonal activation in chronic synovial tissue inflammation.

Abstract The chemokine interleukin-8 (IL-8) is frequently associated with inflammatory diseases, and autoantibodies against IL-8 are present in the periphery at elevated levels in such conditions as rheumatoid arthritis (RA). Circulating free anti-IL-8 IgG autoantibodies correlate with inflammatory parameters and disease severity in RA. In this study, correlations were sought between these disease parameters and other antibody subclasses. We assayed IgM, IgA and IgG anti-IL-8 antibodies and IL-8 immunoglobulin immune complexes in the serum of 29 healthy controls and 56 patients with defined RA, and compared the results with clinical and humoral disease parameters. IgG and IgM antibodies directed against IL-8 were present in all samples. In the disease groups, all isotypes of free anti-IL-8 antibodies correlated with increasing humoral disease parameters like CRP and CIC and their related anti-IL-8 immune complexes. Samples which contained high titers of anti-IL-8 antibody subclasses and complexes were RF subclass-positive, while IgM RF-negative sera showed low levels of anti-IL-8 and complexes. Detectable levels of IgG and IgA RF were found in all sera. Patients with extra-articular organ manifestation showed significantly increased free IgA and IgA/IL-8 complexes, with no correlation to the IgA RF titer or IgA hypergamma-globulinemia. The highest titers were seen in two RA cases with vasculitis and in one patient with colitis. Polyclonal activation of the humoral antibody system, which normally precedes the resolution of an inflammatory response, can itself lead to secondary stimulation of inflammatory processes via immune complex formation. In the immune pathology of RA, it degenerates into a persistent chronic inflammation accompanied by progressive joint destruction. The presence of elevated IgA subclass anti-IL-8 autoantibodies in RA patients with extra-articular manifestations suggests these autoantibodies as a clinically useful marker of disease severity and extra-articular manifestations.

The Translocation Inhibitor CAM741 Interferes with Vascular Cell Adhesion Molecule 1 Signal Peptide Insertion at the Translocon

The cyclopeptolide CAM741 selectively inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), a process that is dependent on its signal peptide. In this study we identified the C-terminal (C-) region upstream of the cleavage site of the VCAM1 signal peptide as most critical for inhibition of translocation by CAM741, but full sensitivity to the compound also requires residues of the hydrophobic (h-) region and the first amino acid of the VCAM1 mature domain. The murine VCAM1 signal peptide, which is less susceptible to translocation inhibition by CAM741, can be converted into a fully sensitive signal peptide by two amino acid substitutions identified as critical for compound sensitivity of the human VCAM1 signal peptide. Using cysteine substitutions of non-critical residues in the human VCAM1 signal peptide and chemical cross-linking of targeted short nascent chains we show that, in the presence of CAM741, the N- and C-terminal segments of the VCAM1 signal peptide could be cross-linked to the cytoplasmic tail of Sec61β, indicating altered positioning of the VCAM1 signal peptide relative to this translocon component. Moreover, translocation of a tag fused N-terminal to the VCAM1 signal peptide is selectively inhibited by CAM741. Our data indicate that the compound inhibits translocation of VCAM1 by interfering with correct insertion of its signal peptide into the translocon.

Up-regulation of interleukin-1β-stimulated interleukin-8 in human keratinocytes by nitric oxide

Nitric oxide (NO) is able to regulate the expression of a number of inflammatory mediators. In this study, the effect of NO on the expression of the chemokine interleukin-8 (IL-8) by primary human keratinocytes and the lines KB and HaCaT was examined. Incubation with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) for 24 hr increased IL-8 protein only in HaCaT cells, partly due to the presence of constitutive interleukin-1 (IL-1). However, in combination with IL-1beta, SNAP enhanced both IL-8 mRNA and protein in all three cell types. Transfection of cells with an IL-8 promoter reporter gene construct showed that the effect of NO was at least partly due to transcriptional activation. Despite small variations in the response to NO by the three cell types, these results demonstrate that NO can up-regulate IL-1beta-stimulated IL-8 expression in human keratinocytes. This study provides a regulatory mechanism which may be important in the context of skin inflammation, and supports the role of NO as an inflammatory mediator in the skin.