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1.
Rheumatology (Oxford) ; 43(1): 39-42, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14566030

ABSTRACT

OBJECTIVE: To investigate the effect of targeting the chemotaxis of monocytes and polymorphonuclear monocytes (PMNs) in situ in MRL-Faslpr arthritis. METHODS: MRL-Faslpr mice were injected intradermally with complete Freund's adjuvant and cellular infiltration into the joint was monitored. Once clinical disease developed, the animals received one of three treatments: MCP-1(9-76); MCP-1(9-76) plus Gro-alpha(8-73); or control peptide, MCP-1 Ala. The bimalleolar ankle width was measured for 11 days and histological examination of the joints was then assessed. RESULTS: Cellular infiltration started after the onset of ankle swelling, and increased progressively. The incidence of swelling and the histopathology was reduced after day 6 of treatment in the MCP-1(9-76)-treated mice. Mice treated with the two antagonists MCP-1(9-76) and Gro-alpha(8-73) displayed a further significant reduction in disease parameters. CONCLUSION: Treatment after disease onset with chemotactic antagonists for monocytes and PMNs significantly alleviated both the swelling and the histopathology seen in arthritis, suggesting that chemokine antagonists are an effective anti-inflammatory therapy.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/drug therapy , Chemokines/antagonists & inhibitors , Animals , Arthritis, Experimental/pathology , Chemokine CCL2/therapeutic use , Chemokine CXCL1 , Chemokines/therapeutic use , Chemotactic Factors/therapeutic use , Drug Therapy, Combination , Female , Intercellular Signaling Peptides and Proteins/therapeutic use , Joints/pathology , Male , Mice , Mice, Mutant Strains , Models, Animal , Monocytes/pathology , Neutrophils/pathology , Peptide Fragments/therapeutic use
2.
J Exp Med ; 196(9): 1141-9, 2002 Nov 04.
Article in English | MEDLINE | ID: mdl-12417625

ABSTRACT

Human hematopoietic tissue contains rare stem cells with multilineage reconstituting ability demonstrable in receptive xenogeneic hosts. We now show that within 3 wk nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver cells regenerate near maximum levels of daughter human hematopoietic stem cells (HSCs) able to repopulate secondary NOD/SCID mice. At this time, most of the human HSCs (and other primitive progenitors) are actively proliferating as shown by their sensitivity to treatments that kill cycling cells selectively (e.g., exposure to high specific-activity [(3)H]thymidine in vitro or 5-fluorouracil in vivo). Interestingly, the proliferating human HSCs were rapidly forced into quiescence by in vivo administration of stromal-derived factor-1 (SDF-1) and this was accompanied by a marked increase in the numbers of human HSCs detectable. A similar result was obtained when transforming growth factor-beta was injected, consistent with a reversible change in HSCs engrafting potential linked to changes in their cell cycle status. By 12 wk after transplant, most of the human HSCs had already entered G(o) and treatment with SDF-1 had no effect on their engrafting activity. These findings point to the existence of novel mechanisms by which inhibitors of HSC cycling can regulate the engrafting ability of human HSCs executing self-renewal divisions in vivo.


Subject(s)
Chemokines, CXC/pharmacology , Hematopoietic Stem Cells/cytology , Transforming Growth Factor beta/pharmacology , Animals , Cell Cycle , Cell Division , Cellular Senescence , Chemokine CXCL12 , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/physiology , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Time Factors , Transforming Growth Factor beta1
3.
Biochem J ; 359(Pt 3): 715-20, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11672447

ABSTRACT

The absolute conservation of tryptophan at position 59 in cytochrome c is related to the unique chemical nature of its indole moiety. The indole side chain of Trp-59 possesses three salient features: bulk, hydrophobicity and the ability of its indole nitrogen to act as a hydrogen-bond donor. Crystallographic evidence identifies the indole nitrogen of Trp-59 as having a stabilizing hydrogen-bonding interaction with the buried carboxylate group of haem propionate 7. Side-chain bulk is also likely to be important because a Phe or Leu residue can replace Trp to give an at least partly functional protein, whereas the smaller Gly or Ser cannot. Semisynthetic analogues were designed to test the importance of the side-chain features of tryptophan by using a recently developed method for stereoselective fragment religation in yeast cytochrome c. Three yeast iso-1 cytochrome c analogues were produced in which Trp-59 was replaced by a non-coded amino acid: p-iodophenylalanine, beta-(3-pyridyl)-alanine or beta-(2-naphthyl)-alanine. Replacement of Trp-59 with these non-coded amino acids allows the reasons for its conservation to be analysed, because they vary with respect to size, hydrophobicity and hydrogen-bond potential. Our results show that decreasing the bulk and hydrophobicity of the side chain at position 59 has a profound but different impact on physicochemical and biological parameters from those of abolishing hydrogen-bond donor potential. This suggests that Trp-59 has both a local and a global stability effect by solvating a buried charge and by having a key role in the packing of the cytochrome c hydrophobic core.


Subject(s)
Cytochrome c Group/chemistry , Cytochrome c Group/genetics , Indoles/chemistry , Tryptophan/chemistry , Cytochrome c Group/metabolism , Models, Molecular , Molecular Structure , Oxidation-Reduction , Oxidoreductases/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/enzymology
4.
J Biol Chem ; 276(47): 43503-8, 2001 Nov 23.
Article in English | MEDLINE | ID: mdl-11571304

ABSTRACT

Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.


Subject(s)
Chemokines, CXC/antagonists & inhibitors , Matrix Metalloproteinases/metabolism , Amino Acid Sequence , Binding Sites , Cell Line , Chemokine CXCL12 , Chemotaxis , Enzyme-Linked Immunosorbent Assay , Hemopexin/metabolism , Humans , Hydrolysis , Protein Binding , Proteoglycans/metabolism
5.
J Exp Med ; 194(6): 855-61, 2001 Sep 17.
Article in English | MEDLINE | ID: mdl-11561000

ABSTRACT

Although numerous chemokines act on monocytes, none of them is specific for these cells. Here, we show that breast and kidney-expressed chemokine (BRAK) is a highly selective monocyte chemoattractant. Migration efficacy and Bordetella pertussis toxin-sensitive Ca(2+) mobilization responses to BRAK were strongly enhanced after treatment of monocytes with the cyclic AMP-elevating agents prostaglandin E(2) and forskolin. BRAK is the first monocyte-selective chemokine, as other types of blood leukocytes or monocyte-derived dendritic cells and macrophages did not respond. Expression in normal skin keratinocytes and dermal fibroblasts as well as lamina propria cells in normal intestinal tissues suggests a homeostatic rather than an inflammatory function for this chemokine. In addition, macrophages were frequently found to colocalize with BRAK-producing fibroblasts. We propose that BRAK is involved in the generation of tissue macrophages by recruiting extravasated precursors to fibroblasts, which are known to secrete essential cytokines for macrophage development.


Subject(s)
Chemokines, CXC/physiology , Macrophages/cytology , Monocytes/metabolism , Cell Differentiation , Cell Movement , Cells, Cultured , Chemokines, CXC/genetics , Cyclic AMP/metabolism , Humans , Intestinal Mucosa/metabolism , Kidney/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/physiology , Monocytes/cytology , Monocytes/drug effects , Monocytes/physiology , Pertussis Toxin , Receptors, Chemokine/metabolism , Signal Transduction , Tissue Distribution , Virulence Factors, Bordetella/metabolism , Virulence Factors, Bordetella/pharmacology
6.
J Exp Med ; 193(12): 1373-81, 2001 Jun 18.
Article in English | MEDLINE | ID: mdl-11413192

ABSTRACT

The T helper (Th) cell pool is composed of specialized cells with heterogeneous effector functions. Apart from Th1 and 2 cells, CXCR5+ T cells have been suggested to be another type of effector T cell specialized for B cell help. We show here that CXCR5+ T cells are heterogeneous, and we identify subsets of CXCR5+ CD4 T cells that differ in function and microenvironmental localization in secondary lymphoid tissues. CD57+CXCR5 T cells, hereafter termed germinal center Th (GC-Th) cells, are localized only in GCs, lack CCR7, and are highly responsive to the follicular chemokine B lymphocyte chemoattractant but not to the T cell zone EBI1-ligand chemokine. Importantly, GC-Th cells are much more efficient than CD57-CXCR5+ T cells or CXCR5- T cells in inducing antibody production from B cells. Consistent with their function, GC-Th cells produce elevated levels of interleukin 10 upon stimulation which, with other cytokines and costimulatory molecules, may help confer their B cell helper activity. Our results demonstrate that CXCR5+ T cells are functionally heterogeneous and that the GC-Th cells, a small subset of CXCR5+ T cells, are the key helpers for B cell differentiation and antibody production in lymphoid tissues.


Subject(s)
B-Lymphocytes/immunology , Germinal Center/immunology , Lymphocyte Cooperation , Receptors, Cytokine/analysis , T-Lymphocytes, Helper-Inducer/immunology , Biomarkers/analysis , Blood/immunology , CD57 Antigens/analysis , Cells, Cultured , Chemokine CCL19 , Chemokine CXCL13 , Chemokines, CC/pharmacology , Chemokines, CXC/pharmacology , Chemotaxis, Leukocyte , Child , Child, Preschool , Cytokines/biosynthesis , Humans , Immunoglobulins/biosynthesis , Immunophenotyping , Palatine Tonsil/immunology , Receptors, CXCR5 , Receptors, Chemokine , T-Lymphocyte Subsets/classification , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Helper-Inducer/drug effects
7.
J Leukoc Biol ; 69(6): 881-4, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11404371

ABSTRACT

Since the discovery of interleukin-8, about 50 chemokines have been identified and characterized. Originally, they were considered as inducible mediators of inflammation, but in recent years, several chemokines were identified that are expressed constitutively and function in physiological traffic and homing of leukocyte-lymphocytes in particular. All chemokines act via seven-transmembrane domain, G protein-coupled receptors. Eighteen such receptors have been identified so far. Studies on structure-activity relationships indicate that chemokines have two main sites of interaction with their receptors, the flexible NH2-terminal region and the conformationally rigid loop that follows the second cysteine. Chemokines are thought to dock onto receptors by means of the loop region, and this contact is believed to facilitate the binding of the NH2-terminal region that results in receptor activation. These studies have also highlighted the importance of the NH2-terminal region for agonistic and antagonistic activity. Recently, we have shown that some naturally occurring chemokines can function as receptor antagonists. These observations suggest a new mechanism for the regulation of leukocyte recruitment during inflammatory and immune reactions, which are based on the combination of agonistic and antagonistic effects.


Subject(s)
Chemokines/physiology , Chemotaxis, Leukocyte/physiology , Receptors, Chemokine/physiology , Amino Acid Motifs , Chemokines/chemistry , Chemokines/pharmacology , Chemotaxis, Leukocyte/drug effects , GTP-Binding Proteins/physiology , Humans , Leukocytes/cytology , Leukocytes/drug effects , Models, Biological , Protein Binding , Protein Structure, Tertiary , Receptors, Chemokine/agonists , Receptors, Chemokine/antagonists & inhibitors , Receptors, Chemokine/chemistry , Structure-Activity Relationship
8.
Blood ; 97(7): 1920-4, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11264152

ABSTRACT

Eotaxin is a potent inducer of eosinophil chemotaxis and was considered as a selective ligand of the CC chemokine receptor 3 (CCR3), which is expressed on eosinophils, basophils, and Th2 lymphocytes. This study shows that eotaxin also interacts with CCR2 and CCR5 and can, thus, affect the responses of monocytes, which express both receptors. In human monocytes pretreatment with eotaxin decreased responsiveness to MCP-1, a selective ligand for CCR2, as well as to RANTES and MIP-1 beta, which bind to CCR5. Similar effects were obtained with transfected cells expressing CCR2 or CCR5, but here a difference became apparent: Eotaxin triggered CCR5 at a concentration of 100 nM but not CCR2 even at 1 microM, suggesting an antagonistic effect on this receptor. In agreement with this observation, eotaxin induced internalization of CCR5 but not of CCR2 in human monocytes and transfected cells. Binding studies showed that eotaxin displaces (125) I-MCP-1 from monocytes in a concentration-dependent manner, and functional experiments showed that eotaxin inhibits MCP-1-induced chemotaxis and enzyme release. The results demonstrate that eotaxin is a CCR5 agonist and a CCR2 antagonist. The present findings suggest a role of eotaxin in the fine-tuning of cellular responses occurring at sites of allergic inflammation, in which both MCP-1 and eotaxin are produced. (Blood. 2001;97:1920-1924)


Subject(s)
Chemokines, CC , Cytokines/physiology , Monocytes/drug effects , Receptors, CCR5/agonists , Receptors, Chemokine/antagonists & inhibitors , Calcium Signaling/drug effects , Chemokine CCL11 , Chemokine CCL2/pharmacology , Chemokine CCL4 , Chemokine CCL5/pharmacology , Chemotaxis, Leukocyte/drug effects , Cytokines/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Endocytosis/drug effects , Eosinophilia/physiopathology , Humans , Hypersensitivity/physiopathology , Inflammation/physiopathology , Macrophage Inflammatory Proteins/pharmacology , Receptors, CCR2 , Transfection
9.
Eur J Immunol ; 31(3): 699-707, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11241273

ABSTRACT

The CXC chemokine stromal cell-derived factor (SDF)-1 is produced constitutively in different tissues. It is the only known ligand for CXCR4, which is widely expressed in leukocytes and in some tissue cells, and acts as coreceptor for X4 HIV strains. Because of the general interest in the mechanisms that regulate the activity of constitutively expressed chemokines, we have studied the inactivation of SDF-1 in cells that bear CXCR4. Here we show that B lymphocytes, NK cells and, to a lesser extent, T lymphocytes inactivate SDF-1 by N-terminal processing. Inactivation is due to cathepsin G which is associated with the membrane of lymphocytes and rapidly cleaves off five N-terminal residues by acting on the Leu(5)-Ser(6) bond yielding SDF-1(6-67). Processing was observed with intact cells, cell membrane preparations and soluble cathepsin G obtained by extraction of the membranes with Triton X-100. Cathepsin G is released by neutrophils and monocytes and binds on the surface of lymphocytes by an apparently saturable process. Analysis of the product obtained, the time course and the sensitivity to inhibitors shows that cathepsin G is the only protease involved. Conversion of SDF-1 to SDF-1(6-67) was complete within minutes to 1-2 h depending on the enzyme source, and was abrogated by inhibitors of serine proteases and chymostatin. Diprotin A, an inhibitor of dipeptidyl peptidase IV, was without effect. Owing to its availability on the surface of SDF-1-responsive cells and its rapid effect, cathepsin G is likely to play a significant role in down-regulating SDF-1 activity.


Subject(s)
Cathepsins/metabolism , Chemokines, CXC/metabolism , Lymphocytes/enzymology , Amino Acid Sequence , B-Lymphocytes/enzymology , Cathepsin G , Cathepsins/drug effects , Cathepsins/isolation & purification , Cells, Cultured , Chemokine CXCL12 , Chemokines, CXC/analysis , Humans , Killer Cells, Natural/enzymology , Membrane Proteins/metabolism , Molecular Sequence Data , Oligopeptides/pharmacology , Peptides/metabolism , Serine Endopeptidases , Serine Proteinase Inhibitors/pharmacology , T-Lymphocytes/enzymology
10.
J Biol Chem ; 276(22): 19356-62, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11279142

ABSTRACT

Resonance energy transfer studies using a pyrene-labeled phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphoglycerol (donor) and the heme (acceptor) of cytochrome c (cyt c) have indicated that ATP causes changes in the conformation of the lipid-bound protein (Rytömaa, M., Mustonen, P., and Kinnunen, P. K. J. (1992) J. Biol. Chem. 267, 22243-22248). Accordingly, after binding cyt c via its so called C-site to neat phosphatidylglycerol liposomes (mole fraction of PG = 1.0) has commenced, further quenching of donor fluorescence is caused by ATP, saturating at 2 mm nucleotide. ATP-induced conformational changes in liposome-associated cyt c could be directly demonstrated by CD in the Soret band region (380-460 nm). The latter data were further supported by time-resolved spectroscopy using the fluorescent cyt c analog with a Zn(2+)-substituted heme moiety. A high affinity ATP-binding site has been demonstrated in cyt c (Craig, D. B., and Wallace, C. J. A. (1993) Protein Sci. 2, 966-976) that is compromised by replacing the invariant Arg(91) to norleucine. Although no major effects on conformation and function of cyt c were concluded due to the modification, a significantly reduced effect by ATP on the lipid-bound [Nle(91)]cyt c was evident, implying that this modulation is mediated via the Arg(91)-containing binding site.


Subject(s)
Adenosine Triphosphate/metabolism , Cytochrome c Group/metabolism , Lipid Metabolism , Animals , Apoptosis , Arginine/chemistry , Binding Sites , Chromatography, High Pressure Liquid , Circular Dichroism , Dose-Response Relationship, Drug , Horses , Hydrogen-Ion Concentration , Liposomes/metabolism , Models, Molecular , Norleucine/chemistry , Protein Binding , Protein Conformation , Spectrometry, Fluorescence , Zinc/chemistry
11.
FEBS Lett ; 489(2-3): 171-5, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11165244

ABSTRACT

Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II that has been shown to be a broad range human chemokine receptor antagonist. Two N-terminal peptides, vMIP-II(1-10) and vMIP-II(1-11)dimer (dimerised through Cys11) were synthesised. Both peptides are shown to bind the CXC chemokine receptor 4 (CXCR4). vMIP-II(1-10) was 1400-fold less potent than the native protein whilst the vMIP-II(1-11)dimer was only 180-fold less potent. In addition, both peptides are CXCR4 antagonists. Through analysis of non-standard, long mixing time two-dimensional nuclear Overhauser enhancement spectroscopy experiments, 13C relaxation data and amide chemical shift temperature gradients for the N-terminus of vMIP-II, we show that this region populates a turn-like structure over residues 5-8, both in the presence and absence of the full protein scaffold. This major conformation is likely to be in fast exchange with other conformational states but it has not previously been detected in monomeric chemokine structures. This and other studies [Elisseeva et al. (2000) J. Biol. Chem. 275, 26799-26805] suggest that there may be a link between the structuring of the short N-terminal chemokine peptides and their ability to bind their receptor.


Subject(s)
Chemokines/chemistry , Peptide Fragments/chemistry , Binding Sites , Binding, Competitive/drug effects , Cell Movement/drug effects , Chemokine CXCL12 , Chemokines/metabolism , Chemokines/pharmacology , Chemokines, CXC/chemistry , Chemokines, CXC/metabolism , Chemokines, CXC/pharmacology , Chemotaxis/drug effects , Dose-Response Relationship, Drug , Humans , Magnetic Resonance Spectroscopy , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Conformation , Receptors, CXCR4/antagonists & inhibitors , Receptors, CXCR4/metabolism , Structure-Activity Relationship , Tumor Cells, Cultured
12.
J Biol Chem ; 276(5): 2986-91, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11110785

ABSTRACT

Th1 and Th2 lymphocytes express a different repertoire of chemokine receptors (CCRs). CXCR3, the receptor for I-TAC (interferon-inducible T cell alpha-chemoattractant), Mig (monokine induced by gamma-interferon), and IP10 (interferon-inducible protein 10), is expressed preferentially on Th1 cells, whereas CCR3, the receptor for eotaxin and several other CC chemokines, is characteristic of Th2 cells. While studying responses that are mediated by these two receptors, we found that the agonists for CXCR3 act as antagonists for CCR3. I-TAC, Mig, and IP10 compete for the binding of eotaxin to CCR3-bearing cells and inhibit migration and Ca(2+) changes induced in such cells by stimulation with eotaxin, eotaxin-2, MCP-2 (monocyte chemottractant protein-2), MCP-3, MCP-4, and RANTES (regulated on activation normal T cell expressed and secreted). A hybrid chemokine generated by substituting the first eight NH(2)-terminal residues of eotaxin with those of I-TAC bound CCR3 with higher affinity than eotaxin or I-TAC (3- and 10-fold, respectively). The hybrid was 5-fold more potent than I-TAC as an inhibitor of eotaxin activity and was effective at concentrations as low as 5 nm. None of the antagonists described induced the internalization of CCR3, indicating that they lack agonistic effects and thus qualify as pure antagonists. These results suggest that chemokines that attract Th1 cells via CXCR3 can concomitantly block the migration of Th2 cells in response to CCR3 ligands, thus enhancing the polarization of T cell recruitment.


Subject(s)
Chemokines, CC , Chemokines, CXC/pharmacology , Intercellular Signaling Peptides and Proteins , Receptors, Chemokine/agonists , Receptors, Chemokine/antagonists & inhibitors , Binding, Competitive , Biological Transport/drug effects , Calcium/metabolism , Chemokine CCL11 , Chemokine CXCL10 , Chemokine CXCL11 , Chemokine CXCL9 , Chemotaxis/drug effects , Cytokines/chemistry , Cytokines/pharmacology , Eosinophils/drug effects , Eosinophils/metabolism , Humans , In Vitro Techniques , Monocyte Chemoattractant Proteins/pharmacology , Receptors, CCR3 , Receptors, CXCR3 , Receptors, Chemokine/metabolism , Th1 Cells/metabolism , Th2 Cells/metabolism
13.
Science ; 289(5482): 1202-6, 2000 Aug 18.
Article in English | MEDLINE | ID: mdl-10947989

ABSTRACT

Tissue degradation by the matrix metalloproteinase gelatinase A is pivotal to inflammation and metastases. Recognizing the catalytic importance of substrate-binding exosites outside the catalytic domain, we screened for extracellular substrates using the gelatinase A hemopexin domain as bait in the yeast two-hybrid system. Monocyte chemoattractant protein-3 (MCP-3) was identified as a physiological substrate of gelatinase A. Cleaved MCP-3 binds to CC-chemokine receptors-1, -2, and -3, but no longer induces calcium fluxes or promotes chemotaxis, and instead acts as a general chemokine antagonist that dampens inflammation. This suggests that matrix metalloproteinases are both effectors and regulators of the inflammatory response.


Subject(s)
Cytokines , Inflammation/metabolism , Matrix Metalloproteinase 2/metabolism , Monocyte Chemoattractant Proteins/metabolism , Animals , Calcium/metabolism , Catalytic Domain , Cell Line , Chemokine CCL7 , Chemokines/antagonists & inhibitors , Chemokines/metabolism , Chemotaxis, Leukocyte , Collagen/metabolism , Enzyme Activation , Gene Library , Hemopexin/chemistry , Hemopexin/metabolism , Humans , Inflammation/pathology , Mass Spectrometry , Matrix Metalloproteinase 2/chemistry , Mice , Protein Binding , Protein Structure, Tertiary , Receptors, Chemokine/antagonists & inhibitors , Receptors, Chemokine/metabolism , Recombinant Proteins/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Two-Hybrid System Techniques
14.
J Exp Med ; 192(3): 313-24, 2000 Aug 07.
Article in English | MEDLINE | ID: mdl-10934220

ABSTRACT

We report that stromal cell-derived factor (SDF)-1 has the remarkable capacity to induce sustained signaling through CXC chemokine receptor 4 (CXCR4). In contrast to other chemokines, such as monocyte chemotactic protein 1 (CC chemokine receptor 2 [CCR2]), macrophage inflammatory protein 1beta (CCR5), liver and activation-regulated chemokine (LARC [CCR6]), Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC [CCR7]), and IP10 (CXCR3), SDF-1 stimulates the prolonged activation of protein kinase B and extracellular signal-regulated kinase (ERK)-2. Activation of protein kinase B is reversed by displacement of SDF-1 from CXCR4 or inhibition of phosphatidylinositol 3-kinase. Although increasing concentrations of SDF-1 enhance CXCR4 internalization, kinase activation is prolonged. In addition, restimulation yields >60% of initial protein kinase B activity, indicating that the remaining receptors are not desensitized. Furthermore, activation is prolonged by inhibiting SDF-1 degradation. The sustained activation of cell survival and mitogenic pathways may account for the unique role of SDF-1 and CXCR4 in embryogenesis and lymphopoiesis.


Subject(s)
Chemokines, CXC/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Receptors, CXCR4/metabolism , Signal Transduction , T-Lymphocytes/metabolism , Androstadienes/pharmacology , Animals , Cells, Cultured , Chemokine CXCL12 , Chemokines, CXC/antagonists & inhibitors , Chemokines, CXC/pharmacology , Dose-Response Relationship, Drug , Down-Regulation , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , Mice , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt , T-Lymphocytes/cytology , Time Factors , Wortmannin
15.
J Biol Chem ; 275(35): 26799-805, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10846182

ABSTRACT

Stromal cell-derived factor 1 (SDF-1), a member of the CXC chemokine family, is the only chemokine to bind to the receptor CXCR4. This receptor is also a co-receptor for syncytia-inducing forms of HIV in CD4(+) cells. In addition, SDF-1 is responsible for attracting mature lymphocytes to the bone marrow and can therefore contribute to host versus graft rejection in bone marrow transplantation. Clearly, by manipulating SDF-1 activity, we could find a possible anti-viral AIDS treatment and aid in bone marrow transplantation. SDF-1 binds to CXCR4 primarily via the N terminus, which appears flexible in the recently determined three-dimensional structure of SDF-1. Strikingly, short N-terminal SDF-1 peptides have been shown to have significant SDF-1 activity. By using NMR, we have determined the major conformation of the N terminus of SDF-1 in a 17-mer (residues 1-17 of SDF-1) and a 9-mer dimer (residues 1-9 of SDF-1 linked by a disulfide bond at residue 9). Residues 5-8 and 11-14 form similar structures that can be characterized as a beta-turn of the beta-alphaR type. These structural motifs are likely to be interconverting with other states, but the major conformation may be important for recognition in receptor binding. These results suggest for the first time that there may be a link between structuring of short N-terminal chemokine peptides and their ability to activate their receptor. These studies will act as a starting point for synthesizing non-peptide analogs that act as CXCR4 antagonists.


Subject(s)
Chemokines, CXC/metabolism , Receptors, Cell Surface/metabolism , Stromal Cells/metabolism , Amino Acid Sequence , Chemokine CXCL12 , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Binding , Protein Conformation
17.
Biochem Cell Biol ; 78(2): 79-86, 2000.
Article in English | MEDLINE | ID: mdl-10874468

ABSTRACT

Many types of physical, spectroscopic, and biological studies of proteins and other macromolecules are facilitated by the incorporation of reporter groups. In many cases these are single atom substitutes, for example isotopes (13C for C), or light (F for H) and heavy (Se for S) atom homologs. In some circumstances the incorporation of two different labels in the same molecule would be greatly desirable. Commonly used protein engineering methods for incorporating them can rarely cope with differential double labeling, and have other limitations such as universal, non-specific, or random incorporation. Although de novo peptide synthesis has the power to achieve highly specific labeling, the difficulties inherent in creating long sequences lead us to propose protein semisynthesis as the most practical approach. By ligating combinations of natural and labeled synthetic fragments to reform holoproteins, we can overcome any of the limitations discussed. Using cytochrome c as a model protein we show that two reporter atoms, selenium and bromine, can be simultaneously and site-specifically incorporated without significant consequences to structure and (or) function. This capability opens up the prospect of advances in a number of areas in structural biology.


Subject(s)
Bromine/metabolism , Cytochrome c Group/metabolism , Diagnostic Imaging , Selenium/metabolism , Animals , Chromatography, Affinity , Chromatography, High Pressure Liquid , Cytochrome c Group/chemistry , Horses , Models, Molecular , Mutagenesis, Site-Directed , Oxidoreductases/metabolism , Peptide Biosynthesis , Selenomethionine/chemistry , Structure-Activity Relationship
18.
Biochemistry ; 39(20): 6053-9, 2000 May 23.
Article in English | MEDLINE | ID: mdl-10821677

ABSTRACT

I-309 is a member of the CC subclass of chemokines and is one of only three human chemokines known to contain an additional, third disulfide bond. The three-dimensional solution structure of I-309 was determined by (1)H nuclear magnetic resonance spectroscopy and dynamic simulated annealing. The structure of I-309, which remains monomeric at high concentrations, was determined on the basis of 978 experimental restraints. The N-terminal region of I-309 was disordered, as has been previously observed for the CC chemokine eotaxin but not others such as MCP-1 and RANTES. This was followed in I-309 by a well-ordered region between residues 13 and 69 that consisted of a 3(10)-helix, a triple-stranded antiparallel beta-sheet, and finally a C-terminal alpha-helix. Root-mean-square deviations of 0.61 and 1.16 were observed for the backbone and heavy atoms, respectively. A comparison of I-309 to eotaxin and HCC-2 revealed a significant structural change in the C-terminal region of the protein. The alpha-helix normally present in chemokines was terminated early and was followed by a short section of extended strand. These changes were a direct result of the additional disulfide bond present in this protein. An examination of the I-309 structure will aid in an understanding of the specificity of this protein with its receptor, CCR8.


Subject(s)
Chemokines, CC/chemistry , Disulfides/chemistry , Monokines , Amino Acid Sequence , Chemokine CCL1 , Crystallography, X-Ray , Dimerization , Humans , Macrophage Inflammatory Proteins , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemistry , Protein Structure, Secondary , Solutions , Structure-Activity Relationship
19.
J Immunol ; 164(7): 3460-4, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10725696

ABSTRACT

A number of orphan G-protein coupled receptors (GPR) have been reported as putative chemokine receptors. One previously reported orphan receptor is an incomplete PCR clone, called GPR2. Here we report the cloning of full-length human (h)GPR2 and mouse (m)GPR2 cDNAs, and the identification of GPR2 as a receptor for a novel CC chemokine called ESkine. hGPR2 is expressed at high levels in testis and small intestine, and at lower levels in other tissues. mGPR2 was expressed at high levels in small intestine, colon, lymph nodes, and Peyer's patches and at lower levels in thymus and spleen. Stimulation of L1.2/hGPR2 transfectants with hESkine induced their migration and resulted in intracellular calcium mobilization. These results provide evidence that GPR2 is a specific receptor for ESkine. We propose that GPR2 be renamed as CCR10. The expression pattern of mGPR2/CCR10 suggests that it may play a role in the homing/trafficking of leukocytes within intestinal and lymphoid environments.


Subject(s)
Chemokines, CC/metabolism , Chemokines/metabolism , GTP-Binding Proteins/metabolism , Receptors, Chemokine/isolation & purification , Amino Acid Sequence , Animals , Calcium/metabolism , Cell Line , Cell Movement/immunology , Chemokine CCL27 , Chemokines/physiology , Cloning, Molecular , DNA, Complementary/isolation & purification , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Organ Specificity/genetics , Organ Specificity/immunology , Receptors, CCR10 , Receptors, Chemokine/biosynthesis , Receptors, Chemokine/genetics , Transfection
20.
J Biol Chem ; 274(47): 33496-503, 1999 Nov 19.
Article in English | MEDLINE | ID: mdl-10559234

ABSTRACT

Using the murine embryonal stem cell system, we have identified a novel gene encoding a highly divergent member of the beta-chemokine family of proinflammatory mediators and have called this protein ESkine. Much of the coding sequence for ESkine overlaps with the 3'-end of a novel interleukin 11 receptor alpha-like sequence on murine chromosome 4. ESkine is produced as two splice variants. One of these variants encodes a classical chemokine with an associated signal peptide, while the other variant (PESKY) possesses the main body of the chemokine but has replaced the signal peptide with an alternative stretch of amino acids that allows for nuclear targeting of this isoform. This differential splicing arises as a result of alternative 5' exon usage. These differentially spliced forms are expressed at discrete tissue loci. Thus, while ESkine is highly expressed in the placenta, PESKY is mainly expressed in the Testes and brain and weakly in the developing embryo. Studies on the proinflammatory properties of ESkine reveal it to be active in inducing polarization of CD4(+) T cells but to be inactive on other hemopoietic cellular populations.


Subject(s)
Alternative Splicing , Cell Nucleus/metabolism , Chemokines, CC/genetics , Chemokines/genetics , Protein Isoforms/genetics , Amino Acid Sequence , Base Sequence , CD4-Positive T-Lymphocytes/cytology , Cell Line , Cell Movement/physiology , Chemokine CCL27 , Chemokines/chemistry , Chemokines/physiology , Chemokines, CC/chemistry , Chemokines, CC/physiology , Humans , Molecular Sequence Data , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism
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