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. 1995 Nov 1;182(5):1301–1314. doi: 10.1084/jem.182.5.1301

Human Mig chemokine: biochemical and functional characterization

PMCID: PMC2192190  PMID: 7595201

Abstract

Mig is a chemokine of the CXC subfamily that was discovered by differential screening of a cDNA library prepared from lymphokine- activated macrophages. The mig gene is inducible in macrophages and in other cells in response to interferon (IFN)-gamma. We have transfected Chinese hamster ovary (CHO) cells with cDNA encoding human Mig and we have derived CHO cell lines from which we have purified recombinant human Mig (rHuMig). rHuMig induced the transient elevation of [Ca2+]i in human tumor-infiltrating T lymphocytes (TIL) and in cultured, activated human peripheral blood-derived lymphocytes. No responses were seen in human neutrophils, monocytes, or Epstein-Barr virus-transformed B lymphoblastoid cell lines. rHuMig was chemotactic for TIL by a modified Boyden chamber assay but rHuMig was not chemotactic for neutrophils or monocytes. The CHO cell lines, IFN-gamma-treated human peripheral-blood monocytes, and IFN-gamma-treated cells of the human monocytic cell line THP-1 all secreted multiple and identical HuMig species as revealed by SDS-PAGE. Using the CHO-derived rHuMig, we have shown that the species' heterogeneity is due to proteolytic cleavage at basic carboxy-terminal residues, and that the proteolysis occurs before and not after rHuMig secretion by the CHO cells. The major species of secreted rHuMig ranged from 78 to 103 amino acids in length, the latter corresponding to the full-length secreted protein predicted from the HuMig cDNA. Carboxy-terminal-truncated forms of rHuMig were of lower specific activity compared to full-length rHuMig in the calcium flux assay, and the truncated species did not block the activity of the full- length species. It is likely that HuMig plays a role in T cell trafficking and perhaps in other aspects of the physiology of activated T cells.

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Selected References

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  1. Angiolillo A. L., Sgadari C., Taub D. D., Liao F., Farber J. M., Maheshwari S., Kleinman H. K., Reaman G. H., Tosato G. Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo. J Exp Med. 1995 Jul 1;182(1):155–162. doi: 10.1084/jem.182.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Averbook B. J., Wei J. P., Perry-Lalley D. M., Rosenberg S. A., Yang J. C. A tumor-elaborated supernatant factor chemotactic for IL-2 expanded tumor infiltrating T-lymphocytes. Lymphokine Cytokine Res. 1993 Feb;12(1):1–8. [PubMed] [Google Scholar]
  3. Ayesh S. K., Azar Y., Babior B. M., Matzner Y. Inactivation of interleukin-8 by the C5a-inactivating protease from serosal fluid. Blood. 1993 Mar 15;81(6):1424–1427. [PubMed] [Google Scholar]
  4. Baggiolini M., Dahinden C. A. CC chemokines in allergic inflammation. Immunol Today. 1994 Mar;15(3):127–133. doi: 10.1016/0167-5699(94)90156-2. [DOI] [PubMed] [Google Scholar]
  5. Balentien E., Han J. H., Thomas H. G., Wen D. Z., Samantha A. K., Zachariae C. O., Griffin P. R., Brachmann R., Wong W. L., Matsushima K. Recombinant expression, biochemical characterization, and biological activities of the human MGSA/gro protein. Biochemistry. 1990 Nov 6;29(44):10225–10233. doi: 10.1021/bi00496a011. [DOI] [PubMed] [Google Scholar]
  6. Brandt E., Petersen F., Flad H. D. A novel molecular variant of the neutrophil-activating peptide NAP-2 with enhanced biological activity is truncated at the C-terminus: identification by antibodies with defined epitope specificity. Mol Immunol. 1993 Aug;30(11):979–991. doi: 10.1016/0161-5890(93)90123-s. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Carr M. W., Roth S. J., Luther E., Rose S. S., Springer T. A. Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3652–3656. doi: 10.1073/pnas.91.9.3652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  10. Clark-Lewis I., Schumacher C., Baggiolini M., Moser B. Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities. J Biol Chem. 1991 Dec 5;266(34):23128–23134. [PubMed] [Google Scholar]
  11. Clore G. M., Appella E., Yamada M., Matsushima K., Gronenborn A. M. Determination of the secondary structure of interleukin-8 by nuclear magnetic resonance spectroscopy. J Biol Chem. 1989 Nov 15;264(32):18907–18911. [PubMed] [Google Scholar]
  12. Cruikshank W. W., Center D. M., Nisar N., Wu M., Natke B., Theodore A. C., Kornfeld H. Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biologic function with CD4 expression. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5109–5113. doi: 10.1073/pnas.91.11.5109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Curling E. M., Hayter P. M., Baines A. J., Bull A. T., Gull K., Strange P. G., Jenkins N. Recombinant human interferon-gamma. Differences in glycosylation and proteolytic processing lead to heterogeneity in batch culture. Biochem J. 1990 Dec 1;272(2):333–337. doi: 10.1042/bj2720333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Denison M. R., Zoltick P. W., Hughes S. A., Giangreco B., Olson A. L., Perlman S., Leibowitz J. L., Weiss S. R. Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events. Virology. 1992 Jul;189(1):274–284. doi: 10.1016/0042-6822(92)90703-R. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Didsbury J. R., Uhing R. J., Tomhave E., Gerard C., Gerard N., Snyderman R. Receptor class desensitization of leukocyte chemoattractant receptors. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11564–11568. doi: 10.1073/pnas.88.24.11564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ernst C. A., Zhang Y. J., Hancock P. R., Rutledge B. J., Corless C. L., Rollins B. J. Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains. J Immunol. 1994 Apr 1;152(7):3541–3549. [PubMed] [Google Scholar]
  17. Falk W., Goodwin R. H., Jr, Leonard E. J. A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods. 1980;33(3):239–247. doi: 10.1016/0022-1759(80)90211-2. [DOI] [PubMed] [Google Scholar]
  18. Farber J. M. A macrophage mRNA selectively induced by gamma-interferon encodes a member of the platelet factor 4 family of cytokines. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5238–5242. doi: 10.1073/pnas.87.14.5238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Farber J. M. HuMig: a new human member of the chemokine family of cytokines. Biochem Biophys Res Commun. 1993 Apr 15;192(1):223–230. doi: 10.1006/bbrc.1993.1403. [DOI] [PubMed] [Google Scholar]
  20. Gao J. L., Becker E. L., Freer R. J., Muthukumaraswamy N., Murphy P. M. A high potency nonformylated peptide agonist for the phagocyte N-formylpeptide chemotactic receptor. J Exp Med. 1994 Dec 1;180(6):2191–2197. doi: 10.1084/jem.180.6.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Grinde B., Seglen P. O. Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes. Biochim Biophys Acta. 1980 Sep 17;632(1):73–86. doi: 10.1016/0304-4165(80)90250-0. [DOI] [PubMed] [Google Scholar]
  24. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  25. Hébert C. A., Vitangcol R. V., Baker J. B. Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. J Biol Chem. 1991 Oct 5;266(28):18989–18994. [PubMed] [Google Scholar]
  26. Imboden J. B., Weiss A., Stobo J. D. The antigen receptor on a human T cell line initiates activation by increasing cytoplasmic free calcium. J Immunol. 1985 Feb;134(2):663–665. [PubMed] [Google Scholar]
  27. Issekutz T. B., Stoltz J. M., vd Meide P. Lymphocyte recruitment in delayed-type hypersensitivity. The role of IFN-gamma. J Immunol. 1988 May 1;140(9):2989–2993. [PubMed] [Google Scholar]
  28. Kelner G. S., Kennedy J., Bacon K. B., Kleyensteuber S., Largaespada D. A., Jenkins N. A., Copeland N. G., Bazan J. F., Moore K. W., Schall T. J. Lymphotactin: a cytokine that represents a new class of chemokine. Science. 1994 Nov 25;266(5189):1395–1399. doi: 10.1126/science.7973732. [DOI] [PubMed] [Google Scholar]
  29. Koch A. E., Polverini P. J., Kunkel S. L., Harlow L. A., DiPietro L. A., Elner V. M., Elner S. G., Strieter R. M. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science. 1992 Dec 11;258(5089):1798–1801. doi: 10.1126/science.1281554. [DOI] [PubMed] [Google Scholar]
  30. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  31. Lee S. J., Nathans D. Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors. J Biol Chem. 1988 Mar 5;263(7):3521–3527. [PubMed] [Google Scholar]
  32. Loscalzo J., Melnick B., Handin R. I. The interaction of platelet factor four and glycosaminoglycans. Arch Biochem Biophys. 1985 Jul;240(1):446–455. doi: 10.1016/0003-9861(85)90049-9. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. MacArthur H., Walter G. Monoclonal antibodies specific for the carboxy terminus of simian virus 40 large T antigen. J Virol. 1984 Nov;52(2):483–491. doi: 10.1128/jvi.52.2.483-491.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Maione T. E., Gray G. S., Petro J., Hunt A. J., Donner A. L., Bauer S. I., Carson H. F., Sharpe R. J. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science. 1990 Jan 5;247(4938):77–79. doi: 10.1126/science.1688470. [DOI] [PubMed] [Google Scholar]
  36. McConkey D. J., Hartzell P., Amador-Pérez J. F., Orrenius S., Jondal M. Calcium-dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex. J Immunol. 1989 Sep 15;143(6):1801–1806. [PubMed] [Google Scholar]
  37. McConkey D. J., Hartzell P., Nicotera P., Orrenius S. Calcium-activated DNA fragmentation kills immature thymocytes. FASEB J. 1989 May;3(7):1843–1849. doi: 10.1096/fasebj.3.7.2497041. [DOI] [PubMed] [Google Scholar]
  38. Miller M. D., Krangel M. S. Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines. Crit Rev Immunol. 1992;12(1-2):17–46. [PubMed] [Google Scholar]
  39. Murphy P. M. The molecular biology of leukocyte chemoattractant receptors. Annu Rev Immunol. 1994;12:593–633. doi: 10.1146/annurev.iy.12.040194.003113. [DOI] [PubMed] [Google Scholar]
  40. Nisbet-Brown E., Cheung R. K., Lee J. W., Gelfand E. W. Antigen-dependent increase in cytosolic free calcium in specific human T-lymphocyte clones. Nature. 1985 Aug 8;316(6028):545–547. doi: 10.1038/316545a0. [DOI] [PubMed] [Google Scholar]
  41. Richmond A., Balentien E., Thomas H. G., Flaggs G., Barton D. E., Spiess J., Bordoni R., Francke U., Derynck R. Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to beta-thromboglobulin. EMBO J. 1988 Jul;7(7):2025–2033. doi: 10.1002/j.1460-2075.1988.tb03042.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schall T. J., Bacon K., Camp R. D., Kaspari J. W., Goeddel D. V. Human macrophage inflammatory protein alpha (MIP-1 alpha) and MIP-1 beta chemokines attract distinct populations of lymphocytes. J Exp Med. 1993 Jun 1;177(6):1821–1826. doi: 10.1084/jem.177.6.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schall T. J., Bacon K., Toy K. J., Goeddel D. V. Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature. 1990 Oct 18;347(6294):669–671. doi: 10.1038/347669a0. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Smith C. A., Williams G. T., Kingston R., Jenkinson E. J., Owen J. J. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature. 1989 Jan 12;337(6203):181–184. doi: 10.1038/337181a0. [DOI] [PubMed] [Google Scholar]
  46. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  47. Spivak-Kroizman T., Lemmon M. A., Dikic I., Ladbury J. E., Pinchasi D., Huang J., Jaye M., Crumley G., Schlessinger J., Lax I. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell. 1994 Dec 16;79(6):1015–1024. doi: 10.1016/0092-8674(94)90032-9. [DOI] [PubMed] [Google Scholar]
  48. Springer T. A. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994 Jan 28;76(2):301–314. doi: 10.1016/0092-8674(94)90337-9. [DOI] [PubMed] [Google Scholar]
  49. Stanhope P. E., Liu A. Y., Pavlat W., Pitha P. M., Clements M. L., Siliciano R. F. An HIV-1 envelope protein vaccine elicits a functionally complex human CD4+ T cell response that includes cytolytic T lymphocytes. J Immunol. 1993 May 15;150(10):4672–4686. [PubMed] [Google Scholar]
  50. 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]
  51. Tashiro K., Tada H., Heilker R., Shirozu M., Nakano T., Honjo T. Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science. 1993 Jul 30;261(5121):600–603. doi: 10.1126/science.8342023. [DOI] [PubMed] [Google Scholar]
  52. Taub D. D., Conlon K., Lloyd A. R., Oppenheim J. J., Kelvin D. J. Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1 alpha and MIP-1 beta. Science. 1993 Apr 16;260(5106):355–358. doi: 10.1126/science.7682337. [DOI] [PubMed] [Google Scholar]
  53. Taub D. D., Lloyd A. R., Conlon K., Wang J. M., Ortaldo J. R., Harada A., Matsushima K., Kelvin D. J., Oppenheim J. J. Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells. J Exp Med. 1993 Jun 1;177(6):1809–1814. doi: 10.1084/jem.177.6.1809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Van Damme J., Van Beeumen J., Conings R., Decock B., Billiau A. Purification of granulocyte chemotactic peptide/interleukin-8 reveals N-terminal sequence heterogeneity similar to that of beta-thromboglobulin. Eur J Biochem. 1989 May 1;181(2):337–344. doi: 10.1111/j.1432-1033.1989.tb14729.x. [DOI] [PubMed] [Google Scholar]
  55. Vanguri P., Farber J. M. Identification of CRG-2. An interferon-inducible mRNA predicted to encode a murine monokine. J Biol Chem. 1990 Sep 5;265(25):15049–15057. [PubMed] [Google Scholar]
  56. Webb L. M., Ehrengruber M. U., Clark-Lewis I., Baggiolini M., Rot A. Binding to heparan sulfate or heparin enhances neutrophil responses to interleukin 8. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7158–7162. doi: 10.1073/pnas.90.15.7158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weiss A., Littman D. R. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. doi: 10.1016/0092-8674(94)90334-4. [DOI] [PubMed] [Google Scholar]
  58. Wilkinson P. C., Newman I. Identification of IL-8 as a locomotor attractant for activated human lymphocytes in mononuclear cell cultures with anti-CD3 or purified protein derivative of Mycobacterium tuberculosis. J Immunol. 1992 Oct 15;149(8):2689–2694. [PubMed] [Google Scholar]
  59. Wun T. C., Kretzmer K. K., Palmier M. O., Day K. C., Huang M. D., Welsch D. J., Lewis C., Wolfe R. A., Zobel J. F., Lange G. W. Comparison of recombinant tissue factor pathway inhibitors expressed in human SK hepatoma, mouse C127, baby hamster kidney, and Chinese hamster ovary cells. Thromb Haemost. 1992 Jul 6;68(1):54–59. [PubMed] [Google Scholar]
  60. Yannelli J. R. The preparation of effector cells for use in the adoptive cellular immunotherapy of human cancer. J Immunol Methods. 1991 May 17;139(1):1–16. doi: 10.1016/0022-1759(91)90345-g. [DOI] [PubMed] [Google Scholar]
  61. Zachariae C. O. Chemotactic cytokines and inflammation. Biological properties of the lymphocyte and monocyte chemotactic factors ELCF, MCAF and IL-8. Acta Derm Venereol Suppl (Stockh) 1993;181:1–37. [PubMed] [Google Scholar]
  62. Zhirnov O., Bukrinskaya A. G. Nucleoproteins of animal influenza viruses, in contrast to those of human strains, are not cleaved in infected cells. J Gen Virol. 1984 Jun;65(Pt 6):1127–1134. doi: 10.1099/0022-1317-65-6-1127. [DOI] [PubMed] [Google Scholar]
  63. Zhou Z., Kim Y. J., Pollok K., Hurtado J., Lee J. K., Broxmeyer H. E., Kwon B. S. Macrophage inflammatory protein-1 alpha rapidly modulates its receptors and inhibits the anti-CD3 mAb-mediated proliferation of T lymphocytes. J Immunol. 1993 Oct 15;151(8):4333–4341. [PubMed] [Google Scholar]
  64. Zucker M. B., Katz I. R., Thorbecke G. J., Milot D. C., Holt J. Immunoregulatory activity of peptides related to platelet factor 4. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7571–7574. doi: 10.1073/pnas.86.19.7571. [DOI] [PMC free article] [PubMed] [Google Scholar]

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