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. 1993 Sep 1;178(3):1127–1132. doi: 10.1084/jem.178.3.1127

Human interferon-inducible protein 10: expression and purification of recombinant protein demonstrate inhibition of early human hematopoietic progenitors

PMCID: PMC2191155  PMID: 8350051

Abstract

Human interferon-inducible protein 10 (IP-10), a member of the family of the small secreted proteins called intercrine cytokines or chemokines, is secreted by interferon gamma-stimulated T cells, monocytes, endothelial cells, and keratinocytes. We have begun to explore the biological properties of IP-10 by cloning and overexpression in baculovirus and in bacterial protein expression systems. A 9.9-kD protein was secreted by infected insect cells, which on sodium dodecyl sulfate-polyacrilamide gel electrophoresis comigrated with keratinocyte IP-10 and with f(22-98), a bacterial recombinant fragment lacking the signal sequence but containing all other residues of IP-10. All three reacted with antibodies recognizing residues 10-98 (alpha IP-10) and 77-98 of IP-10 (alpha 22), demonstrating that it is secreted by keratinocytes and insect cells after removal of the signal sequence but without proteolysis of the COOH-terminal end. Purified rIP- 10 suppresses in vitro colony formation by early human bone marrow progenitor cells which need r-steel factor (rSLF) and rGM-CSF or rSLF and r-erythropoeitin (rEPO). The inhibition is dose dependent, is complete at concentrations > or = 50 ng/ml, is prevented by preincubation of rIP-10 with alpha IP-10, but not by alpha 22, and is seen with highly purified CD34+ cells, suggesting direct effect of rIP- 10 on the progenitors. Combination of rIP-10 and other chemokines at inactive concentrations inhibited colony formation in a synergistic manner. rIP-10 did not affect colony formation in the absence of any growth factors or in the presence of rEPO or rGM-CSF but in absence of rSLF. The effects of IP-10 may be relevant to normal marrow function and might be harnessed to protect human hematopoietic progenitors from the cytotoxic effects of chemotherapy.

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Selected References

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  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Broxmeyer H. E., Sherry B., Cooper S., Lu L., Maze R., Beckmann M. P., Cerami A., Ralph P. Comparative analysis of the human macrophage inflammatory protein family of cytokines (chemokines) on proliferation of human myeloid progenitor cells. Interacting effects involving suppression, synergistic suppression, and blocking of suppression. J Immunol. 1993 Apr 15;150(8 Pt 1):3448–3458. [PubMed] [Google Scholar]
  3. Broxmeyer H. E., Sherry B., Cooper S., Ruscetti F. W., Williams D. E., Arosio P., Kwon B. S., Cerami A. Macrophage inflammatory protein (MIP)-1 beta abrogates the capacity of MIP-1 alpha to suppress myeloid progenitor cell growth. J Immunol. 1991 Oct 15;147(8):2586–2594. [PubMed] [Google Scholar]
  4. Broxmeyer H. E., Sherry B., Lu L., Cooper S., Oh K. O., Tekamp-Olson P., Kwon B. S., Cerami A. Enhancing and suppressing effects of recombinant murine macrophage inflammatory proteins on colony formation in vitro by bone marrow myeloid progenitor cells. Blood. 1990 Sep 15;76(6):1110–1116. [PubMed] [Google Scholar]
  5. Dewald B., Moser B., Barella L., Schumacher C., Baggiolini M., Clark-Lewis I. IP-10, a gamma-interferon-inducible protein related to interleukin-8, lacks neutrophil activating properties. Immunol Lett. 1992 Mar;32(1):81–84. doi: 10.1016/0165-2478(92)90203-z. [DOI] [PubMed] [Google Scholar]
  6. Dimitriadis G. J. Effect of detergents on antibody-antigen interaction. Anal Biochem. 1979 Oct 1;98(2):445–451. doi: 10.1016/0003-2697(79)90165-9. [DOI] [PubMed] [Google Scholar]
  7. Dunlop D. J., Wright E. G., Lorimore S., Graham G. J., Holyoake T., Kerr D. J., Wolpe S. D., Pragnell I. B. Demonstration of stem cell inhibition and myeloprotective effects of SCI/rhMIP1 alpha in vivo. Blood. 1992 May 1;79(9):2221–2225. [PubMed] [Google Scholar]
  8. Graham G. J., Wright E. G., Hewick R., Wolpe S. D., Wilkie N. M., Donaldson D., Lorimore S., Pragnell I. B. Identification and characterization of an inhibitor of haemopoietic stem cell proliferation. Nature. 1990 Mar 29;344(6265):442–444. doi: 10.1038/344442a0. [DOI] [PubMed] [Google Scholar]
  9. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  10. Irie S. [A highly sensitive silver staining for detection of proteins in polyacrylamide gels (author's transl)]. Seikagaku. 1980;52(6):411–413. [PubMed] [Google Scholar]
  11. Lindley I., Aschauer H., Seifert J. M., Lam C., Brunowsky W., Kownatzki E., Thelen M., Peveri P., Dewald B., von Tscharner V. Synthesis and expression in Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9199–9203. doi: 10.1073/pnas.85.23.9199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lord B. I., Dexter T. M., Clements J. M., Hunter M. A., Gearing A. J. Macrophage-inflammatory protein protects multipotent hematopoietic cells from the cytotoxic effects of hydroxyurea in vivo. Blood. 1992 May 15;79(10):2605–2609. [PubMed] [Google Scholar]
  13. Luster A. D., Ravetch J. V. Biochemical characterization of a gamma interferon-inducible cytokine (IP-10). J Exp Med. 1987 Oct 1;166(4):1084–1097. doi: 10.1084/jem.166.4.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Luster A. D., Unkeless J. C., Ravetch J. V. Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature. 1985 Jun 20;315(6021):672–676. doi: 10.1038/315672a0. [DOI] [PubMed] [Google Scholar]
  15. Matsuura Y., Possee R. D., Overton H. A., Bishop D. H. Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J Gen Virol. 1987 May;68(Pt 5):1233–1250. doi: 10.1099/0022-1317-68-5-1233. [DOI] [PubMed] [Google Scholar]
  16. Maze R., Sherry B., Kwon B. S., Cerami A., Broxmeyer H. E. Myelosuppressive effects in vivo of purified recombinant murine macrophage inflammatory protein-1 alpha. J Immunol. 1992 Aug 1;149(3):1004–1009. [PubMed] [Google Scholar]
  17. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sarris A. H., Harding M. W., Jiang T. R., Aftab D., Handschumacher R. E. Immunofluorescent localization and immunochemical determination of cyclophilin-A with specific rabbit antisera. Transplantation. 1992 Nov;54(5):904–910. doi: 10.1097/00007890-199211000-00026. [DOI] [PubMed] [Google Scholar]
  20. Sarris A. H., Palade G. E. Immunofluorescent detection of erythrocyte sialoglycoprotein antigens on murine erythroid cells. J Cell Biol. 1982 Jun;93(3):591–602. doi: 10.1083/jcb.93.3.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  22. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  23. Tempst P., Riviere L. Examination of automated polypeptide sequencing using standard phenyl isothiocyanate reagent and subpicomole high-performance liquid chromatographic analysis. Anal Biochem. 1989 Dec;183(2):290–300. doi: 10.1016/0003-2697(89)90482-x. [DOI] [PubMed] [Google Scholar]
  24. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wolpe S. D., Cerami A. Macrophage inflammatory proteins 1 and 2: members of a novel superfamily of cytokines. FASEB J. 1989 Dec;3(14):2565–2573. doi: 10.1096/fasebj.3.14.2687068. [DOI] [PubMed] [Google Scholar]

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