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R Horuk

Publications and source records attributed to R Horuk.

At least 37 records · Page 2Linked to original sources
PubMed1999

The discovery and therapeutic applications of CCR1 chemokine receptor antagonists.

Chemokines belong to a large family of chemoattractant molecules involved in the directed migration of immune cells. They achieve their cellular effects by direct interaction with cell surface receptors. One such chemokine receptor is CCR1 which is particularly responsive to the C-C chemokines RANTES and MIP-1alpha. This review seeks to highlight the biology, molecular biology, physiology and pathophysiology of CCR1 and looks at the potential of this receptor as a therapeutic target in autoimmunity and inflammation. In addition, current progress in the discovery and development of CCR1 antagonists is summarized.

Journal Article↗
PubMed1998

A His19 to Ala mutant of melanoma growth-stimulating activity is a partial antagonist of the CXCR2 receptor.

Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.

Amino Acid Substitution↗
PubMed1998

Identification and characterization of small molecule functional antagonists of the CCR1 chemokine receptor.

The CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES (regulated on activation normal T cell expressed) have been implicated in rheumatoid arthritis and multiple sclerosis. Since their effects are mediated through the CCR1 chemokine receptor, we set up a small molecule CCR1 antagonist program to search for inhibitors. Through high capacity screening we discovered a number of 4-hydroxypiperidine compounds with CCR1 antagonist activity and report their synthesis and in vitro pharmacology here. Scatchard analysis of the competition binding data revealed that the compounds had Ki values ranging from 40 to 4000 nM. The pharmacological profile of the most potent member of this series, compound 1 (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitri te), was further evaluated. Compound 1 showed concentration-dependent inhibition of MIP-1alpha-induced extracellular acidification and Ca2+ mobilization demonstrating functional antagonism. When given alone, the compound did not elicit any responses, indicating the absence of intrinsic agonist activity. Compound 1 inhibited MIP-1alpha- and RANTES-induced migration in peripheral blood mononuclear cells in a dose-responsive manner. Selectivity testing against a panel of seven transmembrane domain receptors indicated that compound 1 is inactive on a number of receptors at concentrations up to 10 microM. This is the first description of CCR1 receptor antagonists that may be useful in the treatment of chronic inflammatory diseases involving MIP-1alpha, RANTES, and CCR1.

Arthritis, Rheumatoid↗
PubMed1998

Polyclonal antibody directed against human RANTES ameliorates disease in the Lewis rat adjuvant-induced arthritis model.

Adjuvant-induced arthritis (AIA) is one of many animal models of rheumatoid arthritis, a disease characterized by a T-lymphocyte and macrophage cellular infiltrate. We have characterized the development of this disease model with respect to chemokine expression. Increased levels of two chemokines, RANTES, a T-lymphocyte and monocyte chemo-attractant, and KC a chemoattractant for neutrophils, were found in whole blood and in the joint. Surprisingly, levels of MIP-1alpha, another T-lymphocyte and monocyte chemoattractant, were unchanged throughout the course of the disease in whole blood and only slightly elevated in the joint. RANTES expression plays an important role in the disease since a polyclonal antibody to RANTES greatly ameliorated symptoms in animals induced for AIA and was found to be as efficacious as treatment with indomethacin, a non-steroidal anti inflammatory. Polyclonal antibodies to either MIP-1alpha or KC were ineffective. This is the first report to show the importance of RANTES in the development of AIA.

Animals↗
PubMed1998

Neuronal apoptosis induced by HIV-1 gp120 and the chemokine SDF-1 alpha is mediated by the chemokine receptor CXCR4.

CXCR4, a seven transmembrane domain G-protein-coupled receptor for the Cys-X-Cys class of chemokines, is one of several chemokine receptors that can act as a co-receptor with CD4 for the human immunodeficiency virus (HIV-1) glycoprotein gp120 [1-3]. CXCR4 can mediate the entry of HIV-1 strains that specifically infect T cells, such as the IIB strain (see [4] for review). Recent reports indicate that gp120 can signal through CXCR4 [5] and it has been suggested that signal transduction, mediated by the viral envelope, might influence viral-associated cytopathicity or apoptosis [6]. Neuronal apoptosis is a feature of HIV-1 infection in the brain [7,8], although the exact mechanism is unknown. Here, we address the possible role of CXCR4 in inducing apoptosis using cells of the hNT human neuronal cell line; these cells resemble immature post-mitotic cholinergic neurons and have a number of neuronal characteristics [9-15]. We have previously shown that gp120 from the HIV-1 IIIB strain binds with high affinity to CXCR4 expressed on hNT neurons [15]. We now find that both IIIB gp120 and the Cys-X-Cys chemokine SDF-1 alpha can directly induce apoptosis in hNT neurons in the absence of CD4 and in a dose-dependent manner. To our knowledge, this is the first report of a chemokine and an HIV-1 envelope glycoprotein eliciting apoptotic responses through a chemokine receptor.

Apoptosis↗
PubMed1998

Noncompetitive, chemokine-mediated inhibition of basic fibroblast growth factor-induced endothelial cell proliferation.

The proinflammatory and chemoattractant chemokine interleukin-8 (IL-8) inhibits cell proliferation induced by basic fibroblast growth factor (bFGF) in mouse endothelial cells isolated from subcutaneous sponge implant (sponge-induced mouse endothelial cells) and in bovine aortic endothelial GM 7373 cells. The mechanism of action of IL-8 was investigated in GM 7373 cells. IL-8 did not prevent the binding of bFGF to its tyrosine kinase FGF receptors (FGFRs) nor to cell surface heparan sulfate proteoglycans (HSPGs). A transient interaction of IL-8 with the cell before the addition of the growth factor was sufficient to prevent bFGF activity. The inhibitory activity of IL-8 was abolished by protein kinase C (PKC) inhibitors and was mimicked by the PKC activator 12-O-tetradecanoylphorbol-13-acetate. Accordingly, both IL-8 and 12-O-tetradecanoylphorbol-13-acetate caused a approximately 60% decrease of the binding capacity of GM 7373 cells due to the down-regulation of FGFRs. Several C-X-C and C-C chemokines exerted an inhibitory action on bFGF activity similar to IL-8. Soluble heparin, 6-O-desulfated heparin, N-desulfated heparin, and heparan sulfate but not 2-O-desulfated heparin, chondroitin-4-sulfate, hyaluronic acid, and K5 polysaccharide abrogated IL-8 inhibitory activity consistently with the presence of low affinity, high capacity HSPG-like chemokine-binding sites on GM 7373 cells. Finally, neovascularization induced by bFGF in murine subcutaneous sponge implants was reduced significantly by IL-8. In conclusion, IL-8 inhibits the mitogenic activity exerted by bFGF on cultured endothelial cells by a PKC-dependent, noncompetitive mechanism of action that causes FGFR down-regulation. This activity is shared by several chemokines and requires endothelial cell surface HSPGs. The endothelial cell line utilized in the present study may help to elucidate the complex interplay among chemokines, HSPGs, growth factors, and receptors in endothelial cells.

Animals↗
PubMed1998

Identification and characterization of the CXCR4 chemokine receptor in human T cell lines: ligand binding, biological activity, and HIV-1 infectivity.

The CXCR4 chemokine receptor has been shown to respond to the C-X-C chemokine stromal-derived factor (SDF-1) and has recently been shown to be an important coreceptor for HIV-1 infection. In the present paper we have tested a number of human lymphocyte cell lines, including Jurkat, HUT78, CEM, and Sup-T1 for the presence of CXCR4 receptors. We found that these T cell lines bind SDF-1alpha and SDF-1beta with high affinity. The CXCR4 Ab 12G5 inhibited both SDF-1 binding and HIV-1LAI-mediated fusion of CEM. Scatchard analysis revealed the presence of approximately 150,000 SDF-1alpha-binding sites per cell with a Kd between 5 and 10 nM. Cross-competition experiments using unlabeled SDF-1alpha and SDF-1beta revealed that both chemokines are equally capable of displacing their radiolabeled counterparts. Internalization studies with [125]I-SDF-1alpha revealed that Jurkat cells internalized greater than 90% of the ligand by 2 h at 37 degrees C. SDF-1alpha was also chemotactic for Jurkat cells and caused an increase in the rate of extracellular acidification that was half-maximal at 18 nM SDF-1alpha and could be inhibited by pretreatment with the SDF-1 proteins, pertussis toxin, or the Ab 12G5. Finally, SDF-1alpha also caused an increase in the cytosolic Ca2+ concentration in Sup-T1 cells that was abolished by preincubating the cells with pertussis toxin or PMA and inhibited by the Ab 12G5. This molecular characterization of CXCR4 receptors should prove useful in clarifying receptor interaction with SDF-1 proteins and with HIV-1 glycoprotein, with the ultimate aim of targeting the viral interaction for therapeutic intervention.

Binding Sites↗
PubMed1998

The CC chemokine I-309 inhibits CCR8-dependent infection by diverse HIV-1 strains.

Using a chemokine receptor model based on known receptor sequences, we identified several members of the seven transmembrane domain G-protein superfamily as potential chemokine receptors. The orphan receptor ChemR1, which has recently been shown to be a receptor for the CC chemokine I-309, scored very high in our model. We have confirmed that I-309, but not a number of other chemokines, can induce a transient Ca2+ flux in cells expressing CCR8. In addition, the human erythroleukemic cell line K562 responded chemotactically in a dose-responsive manner to this chemokine. Since several chemokine receptors have been shown to be required as coreceptors for HIV-1 infection, we asked whether human immunodeficiency virus type 1 (HIV-1) could efficiently utilize CCR8. Here we show that the CCR8 receptor can serve as a coreceptor for diverse T-cell tropic, dual-tropic, and macrophage-tropic HIV-1 strains and that I-309 was a potent inhibitor of HIV-1 envelope-mediated cell-cell fusion and virus infection. Furthermore, we show by flow cytometry and immunohistochemistry that antibodies generated against the CCR8 receptor amino-terminal peptide cross-reacted with U-87 MG cells stably expressing CCR8, THP-1 cells, HL-60 cells, and human monocytes, a target cell for HIV-1 infectivity in vivo.

Amino Acid Sequence↗
PubMed1998

Genetic subtype-independent inhibition of human immunodeficiency virus type 1 replication by CC and CXC chemokines.

We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1alpha against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4+ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1beta, MIP-1alpha. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1beta, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1beta, MIP-1alpha.

Anti-HIV Agents↗
PubMed1998

An orphan seven-transmembrane domain receptor expressed widely in the brain functions as a coreceptor for human immunodeficiency virus type 1 and simian immunodeficiency virus.

Both CD4 and an appropriate coreceptor are necessary for infection of cells by human immunodeficiency virus type 1 (HIV-1) and most strains of HIV-2. The chemokine receptors CCR5 and CXCR4 are the major HIV-1 coreceptors, although some virus strains can also utilize alternative coreceptors such as CCR3 to infect cells. In contrast, most if not all simian immunodeficiency virus (SIV) strains use CCR5 as a coreceptor, and many SIV strains can use CCR5 independently of CD4. In addition, several orphan seven-transmembrane receptors which can serve as HIV-1 and SIV coreceptors have been identified. Here we report that APJ, an orphan seven-transmembrane domain receptor with homology to the angiotensin receptor family, functions as a coreceptor for a number of HIV-1 and SIV strains. APJ was expressed widely in the human brain and in NT2N neurons. APJ transcripts were also detected by reverse transcription-PCR in the CD4-positive T-cell line C8166, but not in peripheral blood leukocytes, microglia, phytohemagglutinin (PHA)- or PHA/interleukin-2-stimulated peripheral blood mononuclear cells, monocytes, or monocyte-derived macrophages. The widespread distribution of APJ in the central nervous system coupled with its use as a coreceptor by some HIV-1 strains indicates that it may play a role in neuropathogenesis.

Animals↗
PubMed1997

Regulation of human chemokine receptors CXCR4. Role of phosphorylation in desensitization and internalization.

Members of the chemokine receptor family CCR5 and CXCR4 have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. Here, we investigated the regulation of CXCR4 in rat basophilic leukemia cells (RBL-2H3) stably transfected with wild type (Wt CXCR4) or a cytoplasmic tail deletion mutant (DeltaCyto CXCR4) of CXCR4. The ligand, stromal cell derived factor-1 (SDF-1) stimulated higher G-protein activation, inositol phosphate generation, and a more sustained calcium elevation in cells expressing DeltaCyto CXCR4 relative to Wt CXCR4. SDF-1 and phorbol 12-myristate 13-acetate (PMA), but not a membrane permeable cAMP analog induced rapid phosphorylation as well as desensitization of Wt CXCR4. Phosphorylation of DeltaCyto CXCR4 was not detected under any of these conditions. Despite lack of receptor phosphorylation, calcium mobilization by SDF-1 in DeltaCyto CXCR4 cells was partially desensitized by prior treatment with SDF-1. Of interest, the rapid release of calcium was inhibited without affecting the sustained calcium elevation, indicating independent regulatory pathways for these processes. PMA completely inhibited phosphoinositide hydrolysis and calcium mobilization in Wt CXCR4 but only partially inhibited these responses in DeltaCyto CXCR4. cAMP also partially inhibited these responses in both Wt CXCR4 and DeltaCyto CXCR4. SDF-1, PMA, and cAMP caused phosphorylation of phospholipase Cbeta3 in Wt and DeltaCyto CXCR4 cells. Both SDF-1 as well as PMA induced rapid internalization of Wt CXCR4. SDF-1 but not PMA induced internalization of DeltaCyto CXCR4 albeit at reduced levels relative to Wt CXCR4. These results indicate that signaling and internalization of CXCR4 are regulated by receptor phosphorylation dependent and independent mechanisms. Desensitization of CXCR4 signaling, independent of receptor phosphorylation, appears to be a consequence of the phosphorylation of phospholipase Cbeta3.

Amino Acid Sequence↗
PubMed1997

The CXC chemokine stromal cell-derived factor 1 is not responsible for CD8+ T cell suppression of syncytia-inducing strains of HIV-1.

Primary CD8+ T cells from HIV+ asymptomatics can suppress virus production from CD4(+) T cells acutely infected with either non-syncytia-inducing (NSI) or syncytia-inducing (SI) HIV-1 isolates. NSI strains of HIV-1 predominantly use the CCR5 chemokine receptor as a fusion cofactor, whereas fusion of T cell line-adapted SI isolates is mediated by another chemokine receptor, CXCR4. The CCR5 ligands RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta are HIV-1 suppressive factors secreted by CD8+ cells that inhibit NSI viruses. Recently, the CXC chemokine stromal cell-derived factor 1 (SDF-1) was identified as a ligand for CXCR4 and shown to inhibit SI strains. We speculated that SDF-1 might be an effector molecule for CD8+ suppression of SI isolates and assessed several SDF-1 preparations for inhibition of HIV-1LAI-mediated cell-cell fusion, and examined levels of SDF-1 transcripts in CD8(+) T cells. SDF-1 fusion inhibitory activity correlated with the N terminus, and the alpha and beta forms of SDF-1 exhibited equivalent fusion blocking activity. SDF-1 preparations having the N terminus described by Bleul et al. (Bleul, C.C., Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A. & Springer, T.A. (1996) J. Exp. Med. 184, 1101-1109) readily blocked HIV-1LAI-mediated fusion, whereas forms containing two or three additional N-terminal amino acids lacked this activity despite their ability to bind and/or signal through CXCR4. Though SDF-1 is constitutively expressed in most tissues, CD8 T cells contained extremely low levels of SDF-1 mRNA transcripts (<1 transcript/5,000 cells), and these levels did not correlate with virus suppressive activity. We conclude that suppression of SI strains of HIV-1 by CD8+ T cells is unlikely to involve SDF-1.

CD8 Antigens↗
PubMed1997

Human immunodeficiency virus-1 entry into purified blood dendritic cells through CC and CXC chemokine coreceptors.

Blood dendritic cells (DC) are susceptible to both macrophage (M) and T-cell line (T) tropic human immunodeficiency virus type 1. The CC chemokines RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, eotaxin, and, to a lesser extent, monocyte chemoattractant protein-1 (MCP-1) and MCP-4 blocked entry of M-tropic virus into blood DC. The CXC chemokine, SDF-1, a fusin (CXCR4 chemokine receptor) ligand, and an antifusin antibody inhibited DC entry by T-tropic virus. Purified blood DC contained CCR1, CCR2, CCR3, and CCR5 as well as the CXCR4 chemokine receptor RNA transcripts and high levels of fusin on the cell surface. The coexpression of multiple chemokine receptors offers a molecular mechanism to explain the permissiveness of DC for both M- and T-tropic viruses.

Chemokine CCL11↗
PubMed1997

Expression of chemokine receptors by subsets of neurons in the central nervous system.

IL-8 is expressed by activated and neoplastic astrocytes and enhances the survival of hippocampal neurons in vitro. Since mRNA encoding chemokine receptors have been demonstrated in brain, the expression of chemokine receptors by specific cell types in anatomic regions of the central nervous system (CNS) was investigated. Archival tissues from various regions of the CNS were stained with specific mAbs to the Duffy Ag/receptor for chemokines, a promiscuous receptor that binds selected chemokines; the specific receptor for IL-8 (CXCR1); and the receptor (CXCR2) shared by IL-8 and melanoma growth stimulatory activity. The Duffy Ag/receptor for chemokines was expressed exclusively by Purkinje cells in the cerebellum. Chemokine binding and radioligand cross-linking confirmed the presence of a high affinity, promiscuous chemokine receptor in the cerebellum. Although CXCR1 was not expressed in the CNS, CXCR2 was expressed at high levels by subsets of projection neurons in diverse regions of the brain and spinal cord, including the hippocampus, dentate nucleus, pontine nuclei, locus coeruleus, and paraventricular nucleus, and in the anterior horn, interomediolateral cell column, and Clarke's column of the spinal cord. Fibers that express CXCR2 included those in the superior cerebellar peduncle and the substantia gelatinosa. Immunohistochemical analysis of the involved brain tissues from patients with Alzheimer's disease revealed expression of CXCR2 in the neuritic portion of plaques surrounding deposits of amyloid. These data suggest that chemokines may play a role in reactive processes in normal neuronal function and neurodegenerative disorders.

Antigens, CD↗
PubMed1997

CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons.

BACKGROUND: Chemokines are a family of proteins that chemoattract and activate immune cells by interacting with specific receptors on the surface of their targets. We have shown previously that chemokine receptors including the interleukin-8 receptor B (CXCR2) and the Duffy blood group antigen are expressed on subsets of neurons in various regions of the adult nervous system. RESULTS: Using a combination of immunohistochemical staining and receptor binding studies, we show that hNT cells, which are differentiated human neurons derived from the cell line NTera2, express functional chemokine receptors of the C-X-X and C-C types. These chemokine receptors include CXCR2, CXCR4, CCR1 and CCR5. We demonstrate high-affinity binding of both types of chemokines to hNT neurons and dose-dependent chemotactic responses to these chemokines in differentiated, but no t undifferentiated, NTera 2 cells. In addition, we show that the envelop glycoprotein from the T-cell-tropic human immunodeficiency virus 1 (HIV-1) strain IIIB is a CD4-independent, dose-dependent inhibitor of the binding of stromal cell-derived factor 1 to its receptor, CXCR4. CONCLUSIONS: These data support recent findings that members of the chemokine family, including CCR5 and LESTR/Fusin (CXCR4), function as coreceptors in combination with CD4 for HIV-1 invasion. This is the first report of functional expression of chemokine receptors on human neurons. Furthermore, our studies provide for direct CD4-independent association of the viral envelope protein of the HIV-1 strain III with the chemokine receptor CXCR4.

Adult↗
PubMed1997

The role of CXC chemokines in the regulation of angiogenesis in non-small cell lung cancer.

Angiogenesis is a critical component of tumor biology. In recent years newer techniques of cell and molecular biology have led to important advances in our understanding of this process. The regulation of angiogenesis depends on a balance between the activity of local factors that promote (angiogenic factors) or inhibit (angiostatic factors) neovascularization. Nowhere is this paradigm of a balance more apparent than in the study of tumor-associated angiogenesis. Tumors promote angiogenesis through a combination of overexpression of angiogenic factors and local inhibition of angiostatic factors. This strategy leads to an angiogenic environment that promotes tumor growth and metastases. Our laboratory has focused studies on the role of the CXC chemokine family in the regulation of angiogenesis by non-small cell lung cancer (NSCLC). In this article, we review our findings that the CXC chemokine family is composed of members that are either angiogenic or angiostatic. We have found that in NSCLC an imbalance exists in the expression of these factors that favors tumor-derived angiogenesis, and therefore tumor growth and metastases. Furthermore, when this imbalance is corrected to reduce the presence of angiogenic factors or increase the presence of angiostatic factors, tumor growth and metastases are reduced.

Animals↗