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1.
J Immunol ; 195(1): 227-36, 2015 Jul 01.
Article in English | MEDLINE | ID: mdl-25987741

ABSTRACT

Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus' dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (ß-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis.


Subject(s)
Chemokines, CXC/immunology , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Neutrophils/immunology , Receptors, Interleukin-8A/immunology , Receptors, Interleukin-8B/immunology , Viral Proteins/immunology , Animals , Arrestins/genetics , Arrestins/immunology , Calcium/metabolism , Chemokine CCL22/genetics , Chemokine CCL22/immunology , Chemokines, CXC/genetics , Cytomegalovirus/genetics , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/pathology , Cytomegalovirus Infections/virology , Gene Expression Regulation , Genetic Variation , HEK293 Cells , HL-60 Cells , Host-Pathogen Interactions , Humans , Infant , Neutrophils/pathology , Neutrophils/virology , Primary Cell Culture , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8B/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sf9 Cells , Signal Transduction , Spodoptera , Viral Proteins/genetics , beta-Arrestin 2 , beta-Arrestins
2.
Pharmacol Ther ; 133(1): 1-18, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21839114

ABSTRACT

The traffic of the different types of immune cells is an important aspect in the immune response. Chemokines are soluble peptides that are able to attract cells by interaction with chemokine receptors on their target cells. Several different chemokines and receptors exist enabling the specific trafficking of different immune cells. In chronic inflammatory disorders there is abundance of immune cells present at the inflammatory site. This review focuses on the role of chemokine receptors in chronic inflammatory disorders of the lungs, intestine, joints, skin and nervous system and the potential of targeting these receptors as therapeutic intervention in these disorders.


Subject(s)
Chemokines/immunology , Inflammation/immunology , Receptors, Chemokine/immunology , Animals , Atherosclerosis/immunology , Autoimmune Diseases/immunology , Chronic Disease , Humans , Inflammatory Bowel Diseases/immunology , Lung Diseases/immunology , Psoriasis/immunology
3.
Mol Pharmacol ; 80(6): 1108-18, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21948388

ABSTRACT

We have shown previously that different chemical classes of small-molecule antagonists of the human chemokine CXCR2 receptor interact with distinct binding sites of the receptor. Although an intracellular binding site for diarylurea CXCR2 antagonists, such as N-(2-bromophenyl)-N'-(7-cyano-1H-benzotriazol-4-yl)urea (SB265610), and thiazolopyrimidine compounds was recently mapped by mutagenesis studies, we now report on an imidazolylpyrimidine antagonist binding pocket in the transmembrane domain of CXCR2. Using different CXCR2 orthologs, chimeric proteins, site-directed mutagenesis, and in silico modeling, we have elucidated the binding mode of this antagonist. Our in silico-guided mutagenesis studies indicate that the ligand binding cavity for imidazolylpyrimidine compounds in CXCR2 is located between transmembrane (TM) helices 3 (Phe130(3.36)), 5 (Ser217(5.44), Phe220(5.47)), and 6 (Asn268(6.52), Leu271(6.55)) and suggest that these antagonists enter CXCR2 via the TM5-TM6 interface. It is noteworthy that the same interface is postulated as the ligand entry channel in the opsin receptor and is occupied by lipid molecules in the recently solved crystal structure of the CXCR4 chemokine receptor, suggesting a general ligand entrance mechanism for nonpolar ligands to G protein-coupled receptors. The identification of a novel allosteric binding cavity in the TM domain of CXCR2, in addition to the previously identified intracellular binding site, shows the diversity in ligand recognition mechanisms by this receptor and offers new opportunities for the structure-based design of small allosteric modulators of CXCR2 in the future.


Subject(s)
Receptors, Interleukin-8B/metabolism , Allosteric Site/genetics , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Gorilla gorilla , Humans , Ligands , Macaca mulatta , Molecular Sequence Data , Pan troglodytes , Papio , Pongo pygmaeus , Receptors, Interleukin-8B/genetics , Rod Opsins/genetics , Rod Opsins/metabolism , Species Specificity
4.
Eur J Pharmacol ; 668(3): 428-34, 2011 Oct 15.
Article in English | MEDLINE | ID: mdl-21458443

ABSTRACT

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). In this study, we investigated in more detail the mechanism of action of N-ac-PGP in neutrophilic inflammation. N-ac-PGP was chemotactic for human neutrophils via pertussis toxin sensitive G protein-coupled receptors in vitro and directly activated this cell type, which led to cytosolic calcium mobilization and release of CXCL8. Furthermore, using a selective CXCR2 antagonist confirmed that N-ac-PGP-induced neutrophil chemotaxis is mediated through CXCR2 activation. To determine whether N-ac-PGP was solely responsible for the migration and activation of human neutrophils in vitro and not the released CXCL8 upon stimulation with N-ac-PGP, an antibody directed against CXCL8 was used. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of N-ac-PGP whereas effects of CXCL8 were attenuated. These experiments indicate that N-ac-PGP, in addition to the direct induction of chemotaxis, also directly activates neutrophils to release CXCL8. In vivo, this may lead in the long term to a self-maintaining situation enhanced by both N-ac-PGP and CXCL8, leading to a further increase in neutrophil infiltration and chronic inflammation.


Subject(s)
Chemotaxis, Leukocyte/drug effects , Heterotrimeric GTP-Binding Proteins/metabolism , Interleukin-8/metabolism , Neutrophils/cytology , Neutrophils/drug effects , Oligopeptides/pharmacology , Antibodies/immunology , Calcium/metabolism , Collagen/metabolism , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Interleukin-8/antagonists & inhibitors , Interleukin-8/immunology , Neutrophils/metabolism , Peptide Fragments/pharmacology , Pertussis Toxin/toxicity , Receptors, G-Protein-Coupled/metabolism , Receptors, Interleukin-8B/metabolism
5.
Eur J Pharmacol ; 643(1): 29-33, 2010 Sep 15.
Article in English | MEDLINE | ID: mdl-20599927

ABSTRACT

Neutrophils transmigrate from the blood into inflamed tissue via the interaction of chemokines produced in this tissue with chemokine receptors, such as CXCR1 and CXCR2, that are expressed on the membranes of neutrophils. Subsequently, activation of neutrophils will in turn lead to increased tissue damage and thereby enhanced clinical symptoms of inflammatory diseases like chronic obstructive pulmonary disease, inflammatory bowel disease and psoriasis. Besides chemokines, also the collagen-breakdown product N-acetyl-Proline-Glycine-Proline (N-alpha-PGP) attracts neutrophils. In a recent article (Weathington et al., 2006) it was suggested that N-alpha-PGP exerts its effect via CXCR1 and CXCR2. In this study, we show that N-alpha-PGP, in contrast to CXCL8, does not directly activate or interact with CXCR1 or CXCR2. N-alpha-PGP was not able to displace the radioligand [(125)I]CXCL8 from CXCR1 and CXCR2 expressing HEK293T cells or neutrophils. In addition, N-alpha-PGP did not displace the radioligand [(3)H]-SB265610, a CXCR2 antagonist, from CXCR2 expressing cells. Furthermore, N-alpha-PGP was not able to activate G protein signalling in cells expressing CXCR1 and CXCR2. N-alpha-PGP was also not able to recruit beta-arrestin2, an intracellular scaffolding protein involved in G protein-independent signalling, in cells expressing CXCR2. These studies indicate that N-alpha-PGP is not a ligand of CXCR1 or CXCR2.


Subject(s)
Collagen/metabolism , Neutrophils/drug effects , Oligopeptides/pharmacology , Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/metabolism , Binding, Competitive , Cell Culture Techniques , Cell Line , Chemotaxis/drug effects , Humans , Ligands , Neutrophils/immunology , Neutrophils/metabolism , Radioligand Assay , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8B/genetics , Transfection
6.
J Pharmacol Exp Ther ; 329(2): 783-90, 2009 May.
Article in English | MEDLINE | ID: mdl-19190236

ABSTRACT

The chemokine receptor CXCR2 is involved in different inflammatory diseases, like chronic obstructive pulmonary disease, psoriasis, rheumatoid arthritis, and ulcerative colitis; therefore, it is considered an attractive drug target. Different classes of small CXCR2 antagonists have been developed. In this study, we selected seven CXCR2 antagonists from the diarylurea, imidazolylpyrimide, and thiazolopyrimidine class and studied their mechanisms of action at human CXCR2. All compounds are able to displace (125)I-CXCL8 and inhibit CXCL8-induced beta-arrestin2 recruitment. Detailed studies with representatives of each class showed that these compounds displace and antagonize CXCL8, most probably via a noncompetitive, allosteric mechanism. In addition, we radiolabeled the high-affinity CXCR2 antagonist SB265610 [1-(2-bromophenyl)-3-(4-cyano-1H-benzo[d] [1,2,3]-triazol-7-yl)urea] and subjected [(3)H]SB265610 to a detailed analysis. The binding of this radioligand was saturable and reversible. Using [(3)H]SB265610, we found that compounds of the different chemical classes bind to distinct binding sites. Hence, the use of a radiolabeled low-molecular weight CXCR2 antagonist serves as a tool to investigate the different binding sites of CXCR2 antagonists in more detail.


Subject(s)
Phenylurea Compounds/pharmacology , Receptors, Interleukin-8B/antagonists & inhibitors , Allosteric Site , Animals , Binding, Competitive , COS Cells , Chlorocebus aethiops , Humans , Phenylurea Compounds/chemistry , Protein Binding , Radioligand Assay , Structure-Activity Relationship , Transfection
7.
Biochem J ; 393(Pt 3): 635-43, 2006 Feb 01.
Article in English | MEDLINE | ID: mdl-16190863

ABSTRACT

Enzyme structure and dynamics may play a main role in substrate binding and the subsequent steps in the CYP (cytochrome P450) catalytic cycle. In the present study, changes in the structure of human CYP2D6 upon binding of the substrate are studied using steady-state and time-resolved fluorescence methods, focusing not only on the emission of the tryptophan residues, but also on emission of the substrate. As a substrate, MAMC [7-methoxy-4-(aminomethyl)-coumarin] was selected, a compound exhibiting native fluorescence. As well as the wild-type, the W128F (Trp128-->Phe) mutant of CYP2D6 was studied. After binding, a variety of energy transfer possibilities exist, and molecular dynamics simulations were performed to calculate distances and relative orientations of donors and acceptors. Energy transfer from Trp128 to haem appeared to be important; its emission was related to the shortest of the three average tryptophan fluorescence lifetimes observed for CYP2D6. MAMC to haem energy transfer was very efficient as well: when bound in the active site, the emission of MAMC was fully quenched. Steady-state anisotropy revealed that besides the MAMC in the active site, another 2.4% of MAMC was bound outside of the active site to wild-type CYP2D6. The tryptophan residues in CYP2D6 appeared to be less accessible for the external quenchers iodide and acrylamide in presence of MAMC, indicating a tightening of the enzyme structure upon substrate binding. However, the changes in the overall enzyme structure were not very large, since the emission characteristics of the enzyme were not very different in the presence of MAMC.


Subject(s)
Amino Acid Substitution/genetics , Coumarins/metabolism , Cytochrome P-450 CYP2D6/genetics , Cytochrome P-450 CYP2D6/metabolism , Anisotropy , Computer Simulation , Coumarins/chemistry , Cytochrome P-450 CYP2D6/chemistry , Fluorescence Resonance Energy Transfer , Humans , Models, Molecular , Protein Binding , Protein Conformation , Spectrometry, Fluorescence
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