FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2149993174> ?p ?o ?g. }

• W2149993174 endingPage "23292" @default.
• W2149993174 startingPage "23282" @default.
• W2149993174 abstract "The proliferation of human myeloid progenitor cells is negatively regulated in the presence of certain members of the chemokine family of molecules. This includes interleukin 8 (IL-8) and platelet factor 4 (PF4), which in combination are able to synergize, resulting in cell suppression at very low concentrations of these molecules. A series of PF4 and IL-8 mutant proteins were analyzed in an in vitro colony formation assay for myeloid progenitor cells to assess domains of these proteins that are required for activity. Mutation of either of the two DLQ motifs within PF4 resulted in an inactive protein. Perturbations within the IL-8 dimer interface region also resulted in mutants that were incapable of suppressing colony formation. A class of chimeric mutants consisting of domains of either PF4 and IL-8, Gro-α and PF4, or Gro-β and PF4 were observed to inhibit myeloid cell proliferation at concentrations which were between 500- and 5000-fold lower than either the IL-8 or PF4 wild-type proteins alone. These chimeric mutants possessed activities that were comparable to or better than the activity observed when IL-8 and PF4 were added together in vitro. One of these highly active chimeric proteins was observed to be 1000-fold more active than either IL-8 or PF4 alone in suppressing not only the proliferation but also the cell cycling of myeloid progenitor cells following intravenous injection of the mutant into mice. Examination of additional IL-8-based mutants in the colony formation assay, which centered on the perturbation of the amino-terminal “ELR” motif, resulted in the observation that the highly active IL-8 mutant required both aspartic acid at amino acid residue 4 and either glutamine or asparagine at residue 6. Single mutations at either of these positions resulted in mutants with myelosuppressive activity equivalent to wild-type IL-8. Mutants such as IL-8M1 and IL-8M10 were observed to be significantly reduced in their ability to activate isolated human neutrophils, suggesting that separate mechanisms may exist by which myeloid progenitor cells and neutrophils are affected by chemokines. The proliferation of human myeloid progenitor cells is negatively regulated in the presence of certain members of the chemokine family of molecules. This includes interleukin 8 (IL-8) and platelet factor 4 (PF4), which in combination are able to synergize, resulting in cell suppression at very low concentrations of these molecules. A series of PF4 and IL-8 mutant proteins were analyzed in an in vitro colony formation assay for myeloid progenitor cells to assess domains of these proteins that are required for activity. Mutation of either of the two DLQ motifs within PF4 resulted in an inactive protein. Perturbations within the IL-8 dimer interface region also resulted in mutants that were incapable of suppressing colony formation. A class of chimeric mutants consisting of domains of either PF4 and IL-8, Gro-α and PF4, or Gro-β and PF4 were observed to inhibit myeloid cell proliferation at concentrations which were between 500- and 5000-fold lower than either the IL-8 or PF4 wild-type proteins alone. These chimeric mutants possessed activities that were comparable to or better than the activity observed when IL-8 and PF4 were added together in vitro. One of these highly active chimeric proteins was observed to be 1000-fold more active than either IL-8 or PF4 alone in suppressing not only the proliferation but also the cell cycling of myeloid progenitor cells following intravenous injection of the mutant into mice. Examination of additional IL-8-based mutants in the colony formation assay, which centered on the perturbation of the amino-terminal “ELR” motif, resulted in the observation that the highly active IL-8 mutant required both aspartic acid at amino acid residue 4 and either glutamine or asparagine at residue 6. Single mutations at either of these positions resulted in mutants with myelosuppressive activity equivalent to wild-type IL-8. Mutants such as IL-8M1 and IL-8M10 were observed to be significantly reduced in their ability to activate isolated human neutrophils, suggesting that separate mechanisms may exist by which myeloid progenitor cells and neutrophils are affected by chemokines. Myelopoiesis is a complex, highly regulated process, which is dependent on the action of both positive and negative growth factors to control the proliferation of primitive morphologically indistinct cells from hematopoietic organs to supply functional end-stage blood cells. Factors that stimulate cell growth and differentiation have been well characterized and include the colony-stimulating factors (GM-CSF), 1The abbreviations used are: GM-CSFgranulocyte/macrophage colony-stimulating factorILinterleukinPFplatelet factorMIPmacrophage inflammatory proteinMCPmonocyte chemotactic and activating peptideγIP10γ interferon-inducible protein (molecular weight 10,000)HPLChigh performance liquid chromatographyPBSphosphate-buffered salineCHOChinese hamster ovaryCFUcolony-forming unitBFU-Eburst-forming unit of erythroid progenitor cellsGMgranulocyte/macrophageGEMMmultipotential cells. granulocyte colony-stimulating factor, and macrophage colony-stimulating factor), erythropoietin, some of the interleukin family members (e.g. IL-1, IL-3, IL-4, IL-6, IL-9, IL-11) as well as other cytokines including Steel factor 1-3). A number of suppressor molecules have also been identified. These include E-type prostaglandins, H-ferritin, lactoferrin, interferons, tumor necrosis factors, and transforming growth factor-β (1Broxmeyer H.E. Murphy M.J. Concise Reviews in Clinical and Experimental Hematology. Alpha Medical Press, Dayton, OH1992: 119-147Google Scholar, 2Broxmeyer H.E. Oppenheim J.J. Roseo J.L. Gearing A.J.H. Clinical Applications of Cytokines: Role in Pathogenesis, Diagnosis, and Therapy. Oxford University Press, New York1993: 201-230Google Scholar, 3Broxmeyer H.E. Agarwal B.B. Puri R.K. Human Cytokines: Their Role in Disease of Therapy. Blackwell Scientific Publications, Inc., Cambridge1995Google Scholar). More recently, several members of the chemokine family of proteins including macrophage inflammatory protein-1α (MIP-1α), MIP-2α (Gro-β), interleukin 8 (IL-8), platelet factor 4 (PF4), monocyte chemotactic and activating peptide (MCAF/MCP-1), and γ interferon-inducible protein, molecular weight 10,000 (γIP10), have been demonstrated to possess inhibitory activity toward the proliferation of immature stem/progenitor cells in vitro and in vivo(4Graham G.J. Wright E.G. Hewick R. Wolpe S.D. Wilkie N.M. Donaldson D. Lorimore S. Pragnell I.B. Nature. 1990; 344: 442-444Crossref PubMed Scopus (392) Google Scholar, 5Broxmeyer H.E. Sherry B. Lu L. Cooper S. Oh K.-O. Tekamp-Olson P. Kwon B.S. Cerami A. Blood. 1990; 76: 1110-1116Crossref PubMed Google Scholar, 6Broxmeyer H.E. Sherry B. Cooper S. Ruscetti F.W. Williams D.E. Arosio P. Kwon B.S. Cerami A. J. Immunol. 1991; 147: 2586-2594PubMed Google Scholar, 7Dunlop D.J. Wright E.G. Lorimore S. Graham G.J. Holyoake T. Kerr D.J. Wolpe S.D. Pragnell I.B. Blood. 1992; 79: 2221-2225Crossref PubMed Google Scholar, 8Lord B.I. Dexter T.M. Clements J.M. Hunter M.A. Gearing A.J.H. Blood. 1992; 79: 2605-2609Crossref PubMed Google Scholar, 9Maze R. Sherry B. Kwon B.S. Cerami A. Broxmeyer H.E. J. Immunol. 1992; 149: 1004-1009PubMed Google Scholar, 10Broxmeyer H.E. Sherry B. Cooper S. Lu L. Maze R. Beckmann M.P. Cerami A. Ralph P. J. Immunol. 1993; 150: 3448-3458PubMed Google Scholar, 11Sarris A.H. Broxmeyer H.E. Wirthmueller U. Karasavvas N. Cooper S. Lu L. Krueger J. Ravetch J.V. J. Exp. Med. 1993; 178: 1127-1132Crossref PubMed Scopus (89) Google Scholar, 12Han Z.C. Sensebe L. Abgrall J.F. Briere J. Blood. 1990; 75: 1234-1239Crossref PubMed Google Scholar, 13Gewirtz A.M. Calabretta B. Rucinski B. Niewiarowski S. Xu W.Y. J. Clin. Invest. 1989; 83: 1477-1486Crossref PubMed Scopus (118) Google Scholar). granulocyte/macrophage colony-stimulating factor interleukin platelet factor macrophage inflammatory protein monocyte chemotactic and activating peptide γ interferon-inducible protein (molecular weight 10,000) high performance liquid chromatography phosphate-buffered saline Chinese hamster ovary colony-forming unit burst-forming unit of erythroid progenitor cells granulocyte/macrophage multipotential cells. Chemokines are a family of small inducible proteins possessing structural similarities and high amino acid identities(14Oppenheim J.J. Zachariae C.O. Mukaida N. Matsushima K. Annu. Rev. Immunol. 1991; 9: 617-648Crossref PubMed Scopus (1831) Google Scholar, 15Schall T. Cytokine. 1991; 3: 165-183Crossref PubMed Scopus (640) Google Scholar, 16Taub D.T. Oppenheim J.J. Cytokine. 1993; 5: 175-179Crossref PubMed Scopus (74) Google Scholar). Although activity differences exist between the proteins, all are believed to possess chemoattractant properties for various cell types. The family is subdivided into two groups based on positioning of cysteine residues within the amino-terminal domain. The CXC group (2 cysteines with an intervening amino acid) includes IL-8, Gro-α, Gro-β, NAP-2, PF4, ENA78, and γIP10. The three-dimensional structures of IL-8 and PF4 have been solved and show general structural identity(17Zhang X. Chen L. Bancroft D.P. Lai C.K. Maione T.E. Biochemistry. 1994; 33: 8361-8366Crossref PubMed Scopus (159) Google Scholar, 18Clore G.M. Gronenborn A.M. J. Mol. Biol. 1991; 217: 611-620Crossref PubMed Scopus (53) Google Scholar). Protein family members that possess the amino acid motif “ELR” within the amino terminus have all been observed to elicit potent neutrophil chemoattractant and stimulatory activities. This motif has also been shown to be required for specific interaction with either of the two IL-8 receptor proteins on the surface of neutrophils(19Moser B. Dewald B. Barella L. Schumacher C. Baggiolini M. Clark-Lewis I. J. Biol. Chem. 1993; 268: 7125-7128Abstract Full Text PDF PubMed Google Scholar, 20Hebert C.A. Vitangcol R.V. Baker J.B. J. Biol. Chem. 1991; 266: 18989-18994Abstract Full Text PDF PubMed Google Scholar, 21LaRosa G.J. Thomas K.M. Kaufmann M.E. Mark R. White M. Taylor L. Gray G. Witt D. Navarro J. J. Biol. Chem. 1992; 267: 25402-25406Abstract Full Text PDF PubMed Google Scholar). The remaining members of the CXC subgroup display a more diverse activity profile, weak or no neutrophil chemoattracting activity, and less sequence homology to the ELR motif containing subgroup. Neither PF4 nor γIP10 have demonstrated significant neutrophil-related activities(22Walz A. Dewald B. von Tscharner V. Baggiolini M. J. Exp. Med. 1989; 170: 1745-1750Crossref PubMed Scopus (192) Google Scholar, 23Leonard E.J. Yoshimura T. Rot A. Noer K. Walz A. Baggiolini M. Walz D.A. Goetzl E.J. Castor C.W. J. Leukocyte Biol. 1991; 49: 258-261Crossref PubMed Scopus (49) Google Scholar, 24Deuel T.F. Senior R.M. Chang D. Griffin G.L. Heinrikson R.L. Kaiser E.T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4584-4587Crossref PubMed Scopus (325) Google Scholar, 25Dewald B. Moser B. Barella L. Schumacher C. Baggiolini M. Clark-Lewis I. Immunol. Lett. 1992; 32: 81-83Crossref PubMed Scopus (56) Google Scholar). The other half of the chemokine family is characterized by the CC motif (two adjacent cysteine residues located within the amino terminus) and displays a much more diverse sequence homology and activity profile. CC chemokines act predominantly on monocytes, although basophils, lymphocytes, and eosinophils have also been reported to be target cells for various CC proteins including RANTES, MIP-1α, and MCP-1(26Leonard E.J. Yoshimura T. Immunol. Today. 1990; 11: 97-101Abstract Full Text PDF PubMed Scopus (533) Google Scholar, 27Matsushima K. Larsen C.G. DuBois G.C. Oppenheim J.J. J. Exp. Med. 1989; 169: 1485-1490Crossref PubMed Scopus (654) Google Scholar, 28Schall T.J. Bacon K. Toy K.J. Goeddel D.V. Nature. 1990; 347: 669-671Crossref PubMed Scopus (1279) Google Scholar, 29Kuna P. Reddigari S.R. Rucinski D. Oppenheim J.J. Kaplan A.P. J. Exp. Med. 1992; 175: 489-493Crossref PubMed Scopus (180) Google Scholar). Compared to the CXC family, less is understood regarding domains within the proteins which are required for biological activity. However, recent structural information on MIP-1β should facilitate this understanding(30Lodi P.J. Garrett D.S. Kuszewski J. Tsang M.L.-S. Weatherbee J.A. Leonard W.J. Gronenborn A.M. Clore G.M. Science. 1994; 263: 1762-1767Crossref PubMed Scopus (211) Google Scholar). Activated platelets have been observed to release high concentrations of a high molecular weight proteoglycan complex consisting of chondroitin sulfate and PF4(31Moore S. Pepper D.S. Cash J.D. Biochim. Biophys. Acta. 1975; 379: 370-378Crossref PubMed Scopus (88) Google Scholar). In addition to high affinity binding and neutralization of heparin, PF4 also has been observed to inhibit angiogenesis, inhibit bone resorption, and reverse the immunosuppressive effect of lymphoma cells(32Loscalzo J. Melnick B. Handin R.I. Arch. Biochem. Biophys. 1985; 240: 446-455Crossref PubMed Scopus (103) Google Scholar, 33Rucinski D. Niewiarowski S. James P. Walz D.A. Budzynski A.Z. Blood. 1979; 53: 47-62Crossref PubMed Google Scholar, 34Maione T.E. Gray G.S. Petro J. Hunt A.J. Donner A.L. Bauer S.I. Carson H.F. Sharpe R.J. Science. 1990; 247: 77-79Crossref PubMed Scopus (627) Google Scholar, 35Barone A.D. Ghrayeb J. Hammerling U. Zucker M.B. Thorbecke G.J. J. Biol. Chem. 1988; 263: 8710-8715Abstract Full Text PDF PubMed Google Scholar, 36Horton J.E. Harper J. Harper E. Biochim. Biophys. Acta. 1980; 630: 459-463Crossref PubMed Scopus (22) Google Scholar). IL-8 has been observed to possess potent chemotactic and stimulating properties toward human neutrophils in vitro and has been shown to bind with high affinity to either of the two cloned human IL-8 receptors in vitro. In addition to these activities, IL-8 and PF4, as well as MIP-1α, MCP-1, Gro-β, and γIP10, were all observed to inhibit early myeloid progenitor cell proliferation at equivalent concentrations >25 ng/ml(10Broxmeyer H.E. Sherry B. Cooper S. Lu L. Maze R. Beckmann M.P. Cerami A. Ralph P. J. Immunol. 1993; 150: 3448-3458PubMed Google Scholar, 11Sarris A.H. Broxmeyer H.E. Wirthmueller U. Karasavvas N. Cooper S. Lu L. Krueger J. Ravetch J.V. J. Exp. Med. 1993; 178: 1127-1132Crossref PubMed Scopus (89) Google Scholar). Several members of the chemokine family, including NAP-2, Gro-α, Gro-γ, RANTES, and MIP-1β did not possess any inhibitory activities in this assay. A third group of chemokines including Gro-α and Gro-γ (MIP-2β) blocked the inhibitory activity of IL-8 and PF4(10Broxmeyer H.E. Sherry B. Cooper S. Lu L. Maze R. Beckmann M.P. Cerami A. Ralph P. J. Immunol. 1993; 150: 3448-3458PubMed Google Scholar). Similarly, MIP-1β was observed to inhibit the activity of MIP-1α(6Broxmeyer H.E. Sherry B. Cooper S. Ruscetti F.W. Williams D.E. Arosio P. Kwon B.S. Cerami A. J. Immunol. 1991; 147: 2586-2594PubMed Google Scholar, 10Broxmeyer H.E. Sherry B. Cooper S. Lu L. Maze R. Beckmann M.P. Cerami A. Ralph P. J. Immunol. 1993; 150: 3448-3458PubMed Google Scholar). Combinations of any two of the six active chemokines resulted in a synergistic decrease in the amount of each chemokine needed to inhibit proliferation (0.1 ng/ml of each chemokine), suggesting the possibility of a novel mechanism of action on the progenitors(10Broxmeyer H.E. Sherry B. Cooper S. Lu L. Maze R. Beckmann M.P. Cerami A. Ralph P. J. Immunol. 1993; 150: 3448-3458PubMed Google Scholar, 11Sarris A.H. Broxmeyer H.E. Wirthmueller U. Karasavvas N. Cooper S. Lu L. Krueger J. Ravetch J.V. J. Exp. Med. 1993; 178: 1127-1132Crossref PubMed Scopus (89) Google Scholar). The low concentrations of PF4 and IL-8 required to elicit inhibition suggest the presence of protein-based receptors on the progenitor cells. To address this issue, a series of chimeric IL-8/PF4 mutants were expressed, purified, and tested for inhibitory activity toward immature subsets of myeloid progenitor cells. The synthetic genes for human IL-8, PF4, and related mutants were expressed as non-fusion proteins in Escherichia coli (BL21) cells and grown in a 500-ml shaker flask containing 300 μg/ml kanamycin until an absorbance of 0.6 at 600 nm was reached. Cells were induced with isopropyl-1-thio-β-D-galactopyranoside for 3 h at 37°C, followed by centrifugation at 14,000 × g for 30 min to pellet the cells. The cell paste was resuspended in 20 ml of 1 × phosphate-buffered saline (Life Technologies, Inc.) and sonicated for 3 min at 4°C using a Braun-Sonic model 1510 sonicator at 200 watts. Following lysis, the cell suspension was centrifuged for 30 min at 18,000 × g at 4°C. The precipitate from the centrifugation step was extracted in buffer containing 0.05 M Tris-HCl, pH 8.0, 6 M guanidine HCl, 50 mM dithiothreitol at 25°C for 1 h. The extracted material was then diluted with a 50-fold excess (v/v) of buffer containing 25 mM sodium acetate, pH 4.0, 8 M urea. This material was centrifuged at 14,000 × g and the supernatant filtered through 0.45-μm nitrocellulose filters. The protein was loaded onto an S-Sepharose column equilibrated in 25 mM sodium acetate, pH 4.0, 8 M urea and the column was washed with 25 mM sodium acetate, pH 4.0, to remove the urea. A second wash was performed using buffer containing 25 mM sodium acetate, pH 4.0, 0.5 M NaCl. The protein was then eluted using buffer containing 50 mM Tris-HCl, pH 8.0, 1 M NaCl. Fractions containing the appropriate chemokine protein were subjected to refolding overnight in the presence of 1 mM oxidized, 2 mM reduced glutathione at 25°C. Extent of refolding of the proteins was monitored through Poros analytical chromatography. The reduced protein was observed to elute from the Poros column at a different acetonitrile concentration relative to the refolded species. Refolded fractions were pooled and rechromatographed by C4 semi-preparative reverse phase HPLC using a 0-100% acetonitrile gradient in 0.1% trifluoroacetic acid/H2O. Peak fractions were pooled and lyophilized for concentration determination. Purity was assessed by Coomassie staining of SDS-polyacrylamide gel electrophoresis, analytical C4 reverse phase HPLC, and amino acid analysis. Small scale purifications typically yielded several milligrams of highly purified (>95% purity) material. For isolation of human neutrophils, typically 22.5 ml of human blood was layered over 10 ml of Ficoll 1119 and 10 ml of Ficoll 1077 in a 50-ml polypropylene conical tube. The blood was centrifuged in a tabletop centrifuge for 20 min at 1800 rpm at 8°C. Following centrifugation, the neutrophil layer (located just above the pelleted red blood cell layer) was collected, washed in sterile phosphate-buffered saline (without Ca2+ and Mg2+; Life Technologies, Inc.), and pelleted by centrifugation for 5 min at 1800 rpm at 8°C. The neutrophil fraction, which contains some contaminating red blood cells, was resuspended in 27 ml of sterile H2O, which served to lyse the remaining red blood cells. 3 ml of 10 × phosphate-buffered saline were added to the resuspended cells, which were pelleted by centrifugation at 8°C for 5 min at 1800 rpm. The pelleted neutrophils were resuspended in 10 ml of PBS and counted. Resuspended cells were kept on ice until needed for chemokine-dependent assays. Elastase release from human neutrophils was monitored using the fluorescent substrate MeO-Suc-Ala-Ala-Pro-Val-aminomethylcoumarin as described by Hebert et al.(37Hebert C.A. Luscinskas F.W. Kiely J.-M. Luis E.A. Darbonne W.C. Bennett G.L. Liu C.C. Obin M.S. Gimbrone Jr., M.A. Baker J.B. J. Immunol. 1990; 145: 3033-3040PubMed Google Scholar). Isolated human neutrophils were suspended in PBS buffer containing 0.02 M Na2HPO4, pH 7.4, 0.15 M NaCl, 0.2 M Hepes, 1 mg/ml bovine serum albumin, 5 mM glucose, 5 × 10-3 mg/ml cytochalasin B (5 mg/ml stock in Me2SO; Sigma) at a concentration of 2 × 106 cells/ml. 0.5 ml aliquots of the suspended neutrophils were added to 0.5 ml of the PBS buffer solution. Following incubation, cells were re-equilibrated at 37°C for 15 min. Chemokines at varying concentrations were added to the neutrophils while gently mixing. Following addition of the chemokines, the cells were pelleted and 0.75 ml of the resulting supernatant was added to 2.25 ml of PBS in the presence of 5 × 10-3 mg/ml of the elastase substrate (5 mg/ml stock in Me2SO; Peninsula Laboratories, Inc., Belmont, CA). The samples were incubated for 1 h at 37°C then placed on ice for spectrofluorometric analysis. Samples were excited at 380 nm with emission monitored at 460 nm. The ability of IL-8-derived mutants to elicit chemotaxis of isolated human neutrophils was examined using a 48-well micro chemotaxis chamber with a 5-μm pore size filter (Neuroprobe) as described previously(38Falk W. Goodwin Jr., R.H. Leonard E.J. J. Immunol. Methods. 1980; 33: 239-247Crossref PubMed Scopus (251) Google Scholar). Typically, 50,000 neutrophils were added per well and chemokine concentration was varied. After a 30-min incubation period at 37°C, the upper chamber was removed and cells on the filter from the upper chamber were scraped away. The filter was fixed with 100% ethanol, stained with a solution of 0.5% toluidine blue in 3.7% formaldehyde, and counted at 400× magnification. IL-8 was iodinated as described previously (39Thomas K.M. Taylor L. Navarro J. J. Biol. Chem. 1991; 266: 14839-14841Abstract Full Text PDF PubMed Google Scholar) or purchased from DuPont NEN. A stable transfectant CHO cell line, 4ABCHO33(21LaRosa G.J. Thomas K.M. Kaufmann M.E. Mark R. White M. Taylor L. Gray G. Witt D. Navarro J. J. Biol. Chem. 1992; 267: 25402-25406Abstract Full Text PDF PubMed Google Scholar), expressing human neutrophil IL-8 receptor subtype B (huIL8Rb), was used in binding assays to test mutant chemokine binding. Binding was performed as described in (21LaRosa G.J. Thomas K.M. Kaufmann M.E. Mark R. White M. Taylor L. Gray G. Witt D. Navarro J. J. Biol. Chem. 1992; 267: 25402-25406Abstract Full Text PDF PubMed Google Scholar). As described previously(8Lord B.I. Dexter T.M. Clements J.M. Hunter M.A. Gearing A.J.H. Blood. 1992; 79: 2605-2609Crossref PubMed Google Scholar, 9Maze R. Sherry B. Kwon B.S. Cerami A. Broxmeyer H.E. J. Immunol. 1992; 149: 1004-1009PubMed Google Scholar), 1 × 105 low density (<1.077 g/cm3) normal human bone marrow cells were plated in 0.3% agar culture medium with 10% fetal bovine serum (HyClone, Logan, UT) with 100 units/ml recombinant human (rhu) GM-CSF plus 50 ng/ml rhu Steel factor (Immunex Corp., Seattle, WA) in the absence and presence of rhu chemokines for assessment of CFU-GM. For assessment of CFU-GEMM and BFU-E, cells were grown in 0.9% methylcellulose culture medium in the presence of rhu erythropoietin (1-2 units/ml) in combination with 50 ng/ml rhu Steel factor. Three plates were scored per concentration per experiment for CFU-GM, CFU-GEMM, and BFU-E colonies after incubation at 37°C in lowered (5%) O2 for 14 days. The combination of GM-CSF and Steel factor or erythropoietin and Steel factor allow detection of large colonies (usually >1000 cells/colony) which come from early, more immature subsets of CFU-GM, CFU-GEMM, and BFU-E. Levels of significance were determined using Student's t distribution (two-tailed test). C3H/HeJ and BDF1 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were injected intravenously with 0.2 ml of saline/mouse or the stated amount of chemokine and sacrificed 24 h later. Femoral bone marrow was removed, treated with or without high specific activity tritiated thymidine, and plated in 0.3% agar cultured medium with 10% fetal bovine serum in the presence of 10% v/v pokeweed mitogen mouse spleen cell cultured medium as described previously(9Maze R. Sherry B. Kwon B.S. Cerami A. Broxmeyer H.E. J. Immunol. 1992; 149: 1004-1009PubMed Google Scholar). Colonies (>40 cells/aggregate) were scored after 7 days of incubation. The proportion of progenitors in DNA synthesis (S phase of the cell cycle) was estimated using the high specific activity (20 Ci/mM) tritiated thymidine (50 μCi/ml) (DuPont NEN) kill technique and is based on the calculation in vitro of the reduction in the number of colonies formed after pulse exposure of cells for 20 min to “hot” tritiated thymidine as compared with control (McCoy's medium or a comparable amount of non-radioactive “cold” thymidine). A series of chemokine mutants, based on either PF4 or IL-8 native sequences were constructed to examine the domains of these proteins, which are involved in suppression of myeloid progenitor cell proliferation. Fig. 1shows the amino acid sequences of the mutant proteins, which were expressed, purified, and tested in the neutrophil-based assays as well as the myeloid progenitor colony formation assays. For IL-8-based mutants, emphasis was placed on regions surrounding the amino-terminal ELR domain. Mutations were also clustered in the region involved in IL-8 dimerization. For the PF4-based mutants, changes within the DLQ motif, located within the amino-terminal domain were examined. A second DLQ motif, located proximal to the putative heparin binding domain at a reverse β-turn near the carboxyl-terminal domain was also emphasized for mutation. Previously, several groups have reported that peptides containing this motif displayed activity toward suppression of progenitor cell proliferation(13Gewirtz A.M. Calabretta B. Rucinski B. Niewiarowski S. Xu W.Y. J. Clin. Invest. 1989; 83: 1477-1486Crossref PubMed Scopus (118) Google Scholar, 40Caen J.P. Lebeurier I. Aidoudi S. Chen Y.Z. Amiral J. Han Z.C. Blood. 1993; 82: 162aGoogle Scholar). Native sequence proteins as well as mutants were expressed as nonfusion proteins in E. coli (Novagen, pET system). Expression was induced using isopropyl-1-thio-β-D-galactopyranoside, and protein was initially observed as inclusion bodies following lysis of the cells. Proteins were purified from the inclusion bodies by extraction under reducing and denaturing conditions, ion exchange chromatography, a refolding procedure followed by reverse phase HPLC. Correct refolding of the chemokines was monitored by differential retention times using Poros chromatography. Identification of the purified protein was accomplished using amino acid analysis, mass spectrometry, and amino-terminal sequencing. Purity was determined by analytical reverse phase HPLC, mass spectrometry, and SDS-gel electrophoresis. The ability of each of the chemokine proteins to activate neutrophils was tested using a degranulation assay, which followed chemokine-dependent release of elastase. Fig. 2A shows a summary of the activities of the purified chemokine proteins. IL-8, as well as IL-8M3, M4, M6, M7, and M64 all show significant elastase release activity, although compared to IL-8 wild-type, M4 was less active. The PF4-based mutant, PF4M2, displayed approximately 50% of the activity observed with the native sequence IL-8, demonstrating the requirement of the amino-terminal ELR domain for neutrophil-based activity. Some activity was observed with mutants PF4M1 and IL-8M1 only at concentrations of protein greater than 10-6M (10 μg/ml). As expected, PF4, PF4-412, PF4-413, PF4-414, and PF4-421 showed no neutrophil elastase release activity at any of the concentrations tested. Additional IL-8 mutants, designated IL-8M8 (ELQ), IL-8M9 (DLR), and IL-8M10 (DLN), which were developed to examine in greater detail the requirements surrounding the NH2-terminal ELR motif, were also tested in the elastase release assay in a concentration-dependent manner (Fig. 2B). As anticipated, all three displayed either significantly reduced activity or no ability to elicit degranulation of the isolated human neutrophils. Neither IL-8M8 or IL-8M10 elicited any release of elastase at concentrations as high as 4 × 10-6M (40 μg/ml) and 1.25 × 10-5M (100 μg/ml), respectively. IL-8M9 demonstrated the ability to release elastase, although at concentrations approximately 200-fold greater than for the native sequence IL-8. Perturbation of the ELR motif resulted in profound effects on the ability of these chemokine mutants to function on the neutrophil. The ability of each of these mutant proteins to elicit chemotaxis of neutrophils was also examined. Each chemokine mutant was tested in a concentration-dependent manner for ability to elicit chemotaxis of isolated human neutrophils in a boyden chamber. Each concentration of each mutant was tested in triplicate in two separate experiments. Each data point was read in triplicate as well, for a total of 18 data points/concentration/chemokine. The results obtained demonstrate a direct correlation between the ability of the chemokine mutants to elicit chemotaxis of neutrophils and the ability to cause neutrophil degranulation as exhibited in the elastase release assay results (Fig. 3, A-D). With the exception of PF4M2, none of the PF4-derived mutants displayed any chemotactic activity toward neutrophils. Similarly, with the exception of IL-8M1, which showed substantially reduced activity, all of the IL-8-derived mutants exhibited potent neutrophil chemotactic activity, although some reduction in activity was also observed for IL-8M4, IL-8M64, and IL-8M7. However, this decrease also correlated with the data obtained in the elastase release assay. Binding of the IL-8-derived mutants to CHO cells containing the stably transfected IL-8 receptor subtype B was also performed. The B subtype receptor is able to bind with high affinity to IL-8 as well as other “ELR”-containing chemokines including Gro-α, Gro-β, and NAP-2. As shown in Table 1, competition binding experiments utilizing 125I-labeled IL-8 and unlabeled mutant chemokine competitors demonstrated that each of the proteins that was able to activate the neutrophils was also able to bind to the neutrophil receptors. IL-8M1 and PF4M1, which displ" @default.
• W2149993174 created "2016-06-24" @default.
• W2149993174 creator A5004625716 @default.
• W2149993174 creator A5012208330 @default.
• W2149993174 creator A5035307849 @default.
• W2149993174 creator A5038161052 @default.
• W2149993174 creator A5079167638 @default.
• W2149993174 creator A5088610750 @default.
• W2149993174 date "1995-10-01" @default.
• W2149993174 modified "2023-09-26" @default.
• W2149993174 title "High Activity Suppression of Myeloid Progenitor Proliferation by Chimeric Mutants of Interleukin 8 and Platelet Factor 4" @default.
• W2149993174 cites W1490744061 @default.
• W2149993174 cites W1507669417 @default.
• W2149993174 cites W1519137478 @default.
• W2149993174 cites W1546324634 @default.
• W2149993174 cites W1546819161 @default.
• W2149993174 cites W1558296954 @default.
• W2149993174 cites W1574237079 @default.
• W2149993174 cites W1579558830 @default.
• W2149993174 cites W1595430911 @default.
• W2149993174 cites W1598918842 @default.
• W2149993174 cites W1607234954 @default.
• W2149993174 cites W1640512254 @default.
• W2149993174 cites W1669948635 @default.
• W2149993174 cites W1977408485 @default.
• W2149993174 cites W1978136857 @default.
• W2149993174 cites W1983190072 @default.
• W2149993174 cites W1988290538 @default.
• W2149993174 cites W2010661919 @default.
• W2149993174 cites W2010745090 @default.
• W2149993174 cites W2012285989 @default.
• W2149993174 cites W2022481460 @default.
• W2149993174 cites W2027344845 @default.
• W2149993174 cites W2039137468 @default.
• W2149993174 cites W2042371095 @default.
• W2149993174 cites W2044117176 @default.
• W2149993174 cites W2046954016 @default.
• W2149993174 cites W2050820037 @default.
• W2149993174 cites W2058902088 @default.
• W2149993174 cites W2119291835 @default.
• W2149993174 cites W2124687307 @default.
• W2149993174 cites W2145699974 @default.
• W2149993174 cites W2149268788 @default.
• W2149993174 cites W2161784528 @default.
• W2149993174 cites W2170029743 @default.
• W2149993174 cites W2176220887 @default.
• W2149993174 cites W2235333994 @default.
• W2149993174 cites W2397641428 @default.
• W2149993174 cites W2405884584 @default.
• W2149993174 cites W2416128265 @default.
• W2149993174 cites W4229807851 @default.
• W2149993174 cites W4239357337 @default.
• W2149993174 doi "https://doi.org/10.1074/jbc.270.40.23282" @default.
• W2149993174 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7559482" @default.
• W2149993174 hasPublicationYear "1995" @default.
• W2149993174 type Work @default.
• W2149993174 sameAs 2149993174 @default.
• W2149993174 citedByCount "59" @default.
• W2149993174 countsByYear W21499931742013 @default.
• W2149993174 countsByYear W21499931742014 @default.
• W2149993174 countsByYear W21499931742015 @default.
• W2149993174 countsByYear W21499931742016 @default.
• W2149993174 countsByYear W21499931742017 @default.
• W2149993174 countsByYear W21499931742019 @default.
• W2149993174 countsByYear W21499931742020 @default.
• W2149993174 countsByYear W21499931742022 @default.
• W2149993174 countsByYear W21499931742023 @default.
• W2149993174 crossrefType "journal-article" @default.
• W2149993174 hasAuthorship W2149993174A5004625716 @default.
• W2149993174 hasAuthorship W2149993174A5012208330 @default.
• W2149993174 hasAuthorship W2149993174A5035307849 @default.
• W2149993174 hasAuthorship W2149993174A5038161052 @default.
• W2149993174 hasAuthorship W2149993174A5079167638 @default.
• W2149993174 hasAuthorship W2149993174A5088610750 @default.
• W2149993174 hasBestOaLocation W21499931741 @default.
• W2149993174 hasConcept C104317684 @default.
• W2149993174 hasConcept C131665280 @default.
• W2149993174 hasConcept C143065580 @default.
• W2149993174 hasConcept C15729860 @default.
• W2149993174 hasConcept C170493617 @default.
• W2149993174 hasConcept C185592680 @default.
• W2149993174 hasConcept C201750760 @default.
• W2149993174 hasConcept C203014093 @default.
• W2149993174 hasConcept C2775960820 @default.
• W2149993174 hasConcept C2776090121 @default.
• W2149993174 hasConcept C2779282312 @default.
• W2149993174 hasConcept C28328180 @default.
• W2149993174 hasConcept C2994430510 @default.
• W2149993174 hasConcept C502942594 @default.
• W2149993174 hasConcept C54355233 @default.
• W2149993174 hasConcept C67662055 @default.
• W2149993174 hasConcept C86803240 @default.
• W2149993174 hasConcept C8891405 @default.
• W2149993174 hasConcept C89560881 @default.
• W2149993174 hasConcept C95444343 @default.
• W2149993174 hasConceptScore W2149993174C104317684 @default.
• W2149993174 hasConceptScore W2149993174C131665280 @default.
• W2149993174 hasConceptScore W2149993174C143065580 @default.

• next