Development and characterization of a high-throughput in vitro cord formation model insensitive to VEGF inhibition.

Background: Anti-VEGF therapy reduces tumor blood vessels, however, some vessels always remain. These VEGF insensitive vessels may help support continued tumor growth and metastases. Many in vitro assays examining multiple steps of the angiogenic process have been described, but the majority of these assays are sensitive to VEGF inhibition.

Selective role of PKCbeta enzymatic function in regulating cell survival mediated by B cell antigen receptor cross-linking.

Cross-linking of the B cell antigen receptor (BCR) results in the activation of several protein tyrosine kinases leading to phospholipase C-gamma2-dependent phospholipid hydrolysis and Ca2+ mobilization, followed by activation of the protein kinase C (PKC) family members. Sustained Ca2+ release in B lymphocytes is dependent on the membrane localization and activation of the protein tyrosine kinase BTK. Ca2+ release is a tightly regulated process involving BTK membrane localization through its phosphorylation by PKCbeta.

The human specific CCR1 antagonist CP-481,715 inhibits cell infiltration and inflammatory responses in human CCR1 transgenic mice.

We previously described the in vitro characteristics of the potent and selective CCR1 antagonist, CP-481,715. In addition to being selective for CCR1 vs other chemokine receptors, CP-481,715 is also specific for human CCR1 (hCCR1), preventing its evaluation in classical animal models. To address this, we generated mice whereby murine CCR1 was replaced by hCCR1 (knockin) and used these animals to assess the anti-inflammatory properties of CP-481,715.

Expression of rat I-TAC/CXCL11/SCYA11 during central nervous system inflammation: comparison with other CXCR3 ligands.

The chemokines are a large gene superfamily with critical roles in development and immunity. The chemokine receptor CXCR3 appears to play a major role in the trafficking of activated Th1 lymphocytes. There are at least three major ligands for CXCR3: mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11, and of these three ligands, CXCL11 is the least well-characterized.

CP-481,715, a potent and selective CCR1 antagonist with potential therapeutic implications for inflammatory diseases.

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.

Genomic organization of the CC chemokine mip-3alpha/CCL20/larc/exodus/SCYA20, showing gene structure, splice variants, and chromosome localization.

We describe the genomic organization of a recently identified CC chemokine, MIP3alpha/CCL20 (HGMW-approved symbol SCYA20). The MIP-3alpha/CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISH analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3alpha/CCL20, Ala MIP-3alpha/CCL20 and Ser MIP-3alpha/CCL20, that differ by one amino acid at the predicted signal peptide cleavage site.

Expression of IFN-inducible T cell alpha chemoattractant by human endothelial cells is cyclosporin A-resistant and promotes T cell adhesion: implications for cyclosporin A-resistant immune inflammation.

IFN-inducible T cell alpha chemoattractant (I-TAC) is a recently discovered member of the CXC chemokine family. It is a potent T cell chemoattractant expressed by IFN-gamma-treated astrocytes, monocytes, keratinocytes, bronchial epithelial cells, and neutrophils. In this study, we show that I-TAC is also expressed by IFN-gamma-treated endothelial cells (EC), both at the mRNA and protein levels.

Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells.

Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-gamma, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-gamma-inducible CXC chemokines--IFN-inducible protein 10 (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha chemoattractant (I-TAC)--by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ.

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells.

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B.

Genomic organization and biological characterization of the novel human CC chemokine DC-CK-1/PARC/MIP-4/SCYA18.

The chemokines are a group of chemotactic molecules that appear to regulate the directed movement of white blood cells in vitro and in vivo and may therefore play important roles in inflammation and immunity. The genes encoding the chemokines are clustered in close physical proximity to each other. A large cluster of human CC chemokine genes resides on chromosome 17.

Identification of C-C chemokine receptor 1 (CCR1) as the monocyte hemofiltrate C-C chemokine (HCC)-1 receptor.

Hemofiltrate C-C chemokine (HCC)-1 is a recently cloned C-C chemokine that is structurally similar to macrophage inflammatory protein (MIP)-1alpha. Unlike most chemokines, it is constitutively secreted by tissues and is present at high concentrations in normal human plasma. Also atypical for chemokines, HCC-1 is reported not to be chemotactic for leukocytes.

Induction of acute inflammation in vivo by staphylococcal superantigens. II. Critical role for chemokines, ICAM-1, and TNF-alpha.

Superantigens such as staphylococcal enterotoxin A and B (SEA and SEB) activate the immune system by stimulating a large proportion of T lymphocytes through specific Vbeta regions of the TCR and activating macrophages by binding to MHC class II molecules. While the mechanisms by which superantigens activate T lymphocytes have been elucidated, their role in the generation of local immune responses to bacterial invasion is still unclear. In this study we have examined the ability of the superantigens SEA and SEB to elicit an inflammatory reaction in vivo, in s.

Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3.

Chemokines are essential mediators of normal leukocyte trafficking as well as of leukocyte recruitment during inflammation. We describe here a novel non-ELR CXC chemokine identified through sequence analysis of cDNAs derived from cytokine-activated primary human astrocytes. This novel chemokine, referred to as I-TAC (interferon-inducible T cell alpha chemoattractant), is regulated by interferon (IFN) and has potent chemoattractant activity for interleukin (IL)-2-activated T cells, but not for freshly isolated unstimulated T cells, neutrophils, or monocytes.

Chemokine networks in vivo: involvement of C-X-C and C-C chemokines in neutrophil extravasation in vivo in response to TNF-alpha.

In this study, we have evaluated the role of specific chemotactic cytokines in leukocyte recruitment to s.c. tissue in response to TNF-alpha in vivo.

Cloning, characterization, and mapping of a murine promiscuous chemokine receptor gene: homolog of the human Duffy gene.

We report here the isolation and genomic organization of the orthologous mouse Duffy gene, named Dfy. It is a single copy gene located in chromosome 1 in a region homologous to the human Duffy gene (FY). Sequence analyses indicate that Dfy consists of two exons: exon 1 of 55 nucleotides, which encodes 7 amino acid residues; and exon 2 of 1038 nucleotides, which encodes 327 residues.

Identification of G-protein binding sites of the human interleukin-8 receptors by functional mapping of the intracellular loops.

Interleukin 8 (IL-8) is considered to be a major mediator of the inflammatory response. Recent evidence indicates that a direct physical association occurs between IL-8 receptors and the alpha subunit of guanine nucleotide regulatory protein (Gi(alpha)2) upon stimulation of human neutrophils by IL-8. In the present study, we identified by site-directed mutagenesis key residues within the three intracellular loops of the IL-8RA receptor involved in the interaction with Gi(alpha)2.

Diverging signal transduction pathways activated by interleukin 8 (IL-8) and related chemokines in human neutrophils. IL-8 and Gro-alpha differentially stimulate calcium influx through IL-8 receptors A and B.

Interleukin 8 (IL-8) and Gro-alpha are members of the CXC branch of a family of cytokines recently designated the "chemokine" superfamily. Recent evidence indicates that, contrary to previously held beliefs, IL-8 and Gro-alpha may not be perceived equivalently by neutrophils. In this study, we have evaluated the effects of IL-8 and Gro-alpha on the rate of calcium influx in human neutrophils and in 293 cells transfected with type A or type B IL-8 receptors.

The Duffy antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Duffy negative individuals who lack the erythrocyte receptor.

The Duffy antigen/receptor for chemokines (DARC), first identified on erythrocytes, functions not only as a promiscuous chemokine receptor but also as a receptor for the malarial parasite, Plasmodium vivax. The recent finding that DARC is ubiquitously expressed by endothelial cells lining postcapillary venules provides a possible insight into the function of this receptor because this anatomic site is an active interface for leukocyte trafficking. However, the biological significance of DARC is questionable since it has not yet been determined whether individuals lacking the expression of this protein on their erythrocytes (Duffy negative individuals), who are apparently immunologically normal, express the receptor on endothelial cells.

Functional and biochemical analysis of the cloned Duffy antigen: identity with the red blood cell chemokine receptor.

The Duffy blood group antigen has been postulated to be a receptor on red blood cells (RBCs) for the malarial parasite Plasmodium vivax and a promiscuous receptor for the chemokine superfamily of inflammatory proteins. Recently, the Duffy antigen glycoprotein D cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). We have analyzed the binding properties of the cloned Duffy antigen.

Identification of a promiscuous inflammatory peptide receptor on the surface of red blood cells.

Erythrocytes have long been appreciated as transporters and exchangers of O2 and CO2 between the lungs and the tissues. Here we examine the role of erythrocytes as potential mediators of inflammatory processes by assessing their ability to bind to a number of inflammatory peptides of the chemokine (for chemoattractant cytokine) superfamily. Radiolabeled chemokines of either the C-X-C (IL-8, MGSA/gro, NAP-2) or C-C (RANTES, MCP-1) class bind reversibly to red cell surface receptors numbering 1000-9000 sites/cell with a Kd of approximately 5 nM.

The human erythrocyte inflammatory peptide (chemokine) receptor. Biochemical characterization, solubilization, and development of a binding assay for the soluble receptor.

In addition to the two human interleukin-8 (IL-8) receptors that have been cloned, IL-8RA and IL-8RB, we recently described a binding protein in human erythrocytes that binds IL-8 and monocyte chemotactic peptide-1 (MCP-1), which we have termed the chemokine (CK) receptor. This communication describes the biochemical characterization, detergent solubilization, and development of a solubilized receptor binding assay for the erythrocyte CK receptor. Competitive 125I-IL-8 binding studies in cells transfected with IL-8RA and IL-8RB revealed that only IL-8 and MGSA were able to displace the radiolabeled IL-8 from these cells.

Uncoupling of early signal transduction events from effector function in human peripheral blood neutrophils in response to recombinant macrophage inflammatory proteins-1 alpha and -1 beta.

Macrophage inflammatory proteins-1 (MIP-1) alpha and beta are members of the C-C branch of the platelet factor 4 superfamily of cytokines, recently designated the "chemokine" superfamily. It has been suggested that the major cellular targets for the biologic activities of the C-C chemokines are the mononuclear leukocytes. However, the original designation of murine MIP-1 proteins as inflammatory mediators was based on suggestions that they activated neutrophil functions such as chemotaxis, the respiratory burst, and degranulation.

Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor.

The immunoregulatory proteins C-C chemokines are potent chemoattractants of lymphocytes and monocytes, as well as activators and attractants of eosinophils and basophils. We have isolated a cDNA that encodes a seven transmembrane-spanning receptor, with homology to other chemoattractant receptors, that encodes a protein designated C-C CKR-1 that acts as a receptor for the C-C chemokines. Human and murine macrophage inflammatory protein 1 alpha (MIP-1 alpha), human human monocyte chemotactic protein 1 (MCP-1), and RANTES all bind to the C-C CKR-1 with varying affinities.