Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1994 Jul 15;301(Pt 2):545–550. doi: 10.1042/bj3010545

Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2 alpha and CINC-2 beta, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization.

H Nakagawa 1, N Komorita 1, F Shibata 1, A Ikesue 1, K Konishi 1, M Fujioka 1, H Kato 1
PMCID: PMC1137115  PMID: 8043001

Abstract

Four basic neutrophil chemotactic factors (chemokines) have been purified from conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in the rat. On the basis of their N-terminal amino acid sequences, one of the chemokines was identical with rat GRO/cytokine-induced neutrophil chemoattractant (CINC) which we reported previously, and another was identical with rat macrophage inflammatory protein-2 (MIP-2). Two other chemokines were novel chemoattractants related to MIP-2. The novel chemokines are referred to as rat GRO/CINC-2 alpha and CINC-2 beta, and consequently CINC and rat MIP-2 are renamed rat GRO/CINC-1 and CINC-3 respectively. The complete amino acid sequences of purified CINC-2 alpha and CINC-3 were determined by analysis of the fragments isolated from proteinase V8-treated CINCs. The cDNA for CINC-2 beta was cloned by reverse transcription/PCR amplification using specific primers starting with total RNA extracted from lipopolysaccharide-stimulated rat macrophages. A comparison of the amino acid sequence encoded by the cDNA with the N-terminal amino acid sequence of purified CINC-2 beta revealed that mature CINC-2 beta is a 68-residue chemoattractant produced by cleavage of a 32-residue signal peptide. The difference in amino acid sequences between CINC-2 alpha and CINC-2 beta consisted of only three C-terminal residues. Rat GRO/CINC-2 alpha is a major chemokine, and the four purified chemokines have similar chemotactic activity, suggesting that they contribute to neutrophil infiltration into inflammatory sites in rats.

Full text

PDF
546

Images in this article

547Figure 2
on p.547

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anisowicz A., Bardwell L., Sager R. Constitutive overexpression of a growth-regulated gene in transformed Chinese hamster and human cells. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7188–7192. doi: 10.1073/pnas.84.20.7188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baggiolini M., Clark-Lewis I. Interleukin-8, a chemotactic and inflammatory cytokine. FEBS Lett. 1992 Jul 27;307(1):97–101. doi: 10.1016/0014-5793(92)80909-z. [DOI] [PubMed] [Google Scholar]
  3. Baggiolini M., Walz A., Kunkel S. L. Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils. J Clin Invest. 1989 Oct;84(4):1045–1049. doi: 10.1172/JCI114265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beaubien B. C., Collins P. D., Jose P. J., Totty N. F., Hsuan J., Waterfield M. D., Williams T. J. A novel neutrophil chemoattractant generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to interleukin 8. Biochem J. 1990 Nov 1;271(3):797–801. doi: 10.1042/bj2710797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Begg G. S., Pepper D. S., Chesterman C. N., Morgan F. J. Complete covalent structure of human beta-thromboglobulin. Biochemistry. 1978 May 2;17(9):1739–1744. doi: 10.1021/bi00602a024. [DOI] [PubMed] [Google Scholar]
  6. Fukuhara M., Tsurufuji S. The effect of locally injected anti-inflammatory drugs on the carrageenin granuloma in rats. Biochem Pharmacol. 1969 Feb;18(2):475–484. doi: 10.1016/0006-2952(69)90223-8. [DOI] [PubMed] [Google Scholar]
  7. Goodman R. B., Foster D. C., Mathewes S. L., Osborn S. G., Kuijper J. L., Forstrom J. W., Martin T. R. Molecular cloning of porcine alveolar macrophage-derived neutrophil chemotactic factors I and II; identification of porcine IL-8 and another intercrine-alpha protein. Biochemistry. 1992 Nov 3;31(43):10483–10490. doi: 10.1021/bi00158a011. [DOI] [PubMed] [Google Scholar]
  8. Haskill S., Peace A., Morris J., Sporn S. A., Anisowicz A., Lee S. W., Smith T., Martin G., Ralph P., Sager R. Identification of three related human GRO genes encoding cytokine functions. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7732–7736. doi: 10.1073/pnas.87.19.7732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hirasawa N., Watanabe M., Mue S., Watanabe K., Tsurufuji S., Ohuchi K. Induction of neutrophil infiltration by rat chemotactic cytokine (CINC) and its inhibition by dexamethasone in rats. Inflammation. 1992 Apr;16(2):187–196. doi: 10.1007/BF00918958. [DOI] [PubMed] [Google Scholar]
  10. Huang S., Paulauskis J. D., Godleski J. J., Kobzik L. Expression of macrophage inflammatory protein-2 and KC mRNA in pulmonary inflammation. Am J Pathol. 1992 Oct;141(4):981–988. [PMC free article] [PubMed] [Google Scholar]
  11. Iida M., Watanabe K., Tsurufuji M., Takaishi K., Iizuka Y., Tsurufuji S. Level of neutrophil chemotactic factor CINC/gro, a member of the interleukin-8 family, associated with lipopolysaccharide-induced inflammation in rats. Infect Immun. 1992 Apr;60(4):1268–1272. doi: 10.1128/iai.60.4.1268-1272.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Konishi K., Takata Y., Yamamoto M., Yomogida K., Watanabe K., Tsurufuji S., Fujioka M. Structure of the gene encoding rat neutrophil chemo-attractant Gro. Gene. 1993 Apr 30;126(2):285–286. doi: 10.1016/0378-1119(93)90382-d. [DOI] [PubMed] [Google Scholar]
  13. Matsushima K., Morishita K., Yoshimura T., Lavu S., Kobayashi Y., Lew W., Appella E., Kung H. F., Leonard E. J., Oppenheim J. J. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J Exp Med. 1988 Jun 1;167(6):1883–1893. doi: 10.1084/jem.167.6.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nakagawa H., Ikesue A., Hatakeyama S., Kato H., Gotoda T., Komorita N., Watanabe K., Miyai H. Production of an interleukin-8-like chemokine by cytokine-stimulated rat NRK-49F fibroblasts and its suppression by anti-inflammatory steroids. Biochem Pharmacol. 1993 Apr 6;45(7):1425–1430. doi: 10.1016/0006-2952(93)90041-t. [DOI] [PubMed] [Google Scholar]
  15. Nakagawa H., Ikesue A., Kato H., Debuchi H., Watanabe K., Tsurufuji S., Naganawa M., Mitamura M. Changes in the levels of rat interleukin 8/CINC and gelatinase in the exudate of carrageenin-induced inflammation in rats. J Pharmacobiodyn. 1992 Sep;15(9):461–466. doi: 10.1248/bpb1978.15.461. [DOI] [PubMed] [Google Scholar]
  16. Oppenheim J. J., Zachariae C. O., Mukaida N., Matsushima K. Properties of the novel proinflammatory supergene "intercrine" cytokine family. Annu Rev Immunol. 1991;9:617–648. doi: 10.1146/annurev.iy.09.040191.003153. [DOI] [PubMed] [Google Scholar]
  17. Proost P., Wuyts A., Conings R., Lenaerts J. P., Billiau A., Opdenakker G., Van Damme J. Human and bovine granulocyte chemotactic protein-2: complete amino acid sequence and functional characterization as chemokines. Biochemistry. 1993 Sep 28;32(38):10170–10177. doi: 10.1021/bi00089a037. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Schröder J. M., Mrowietz U., Morita E., Christophers E. Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity. J Immunol. 1987 Nov 15;139(10):3474–3483. [PubMed] [Google Scholar]
  20. Schröder J. M., Persoon N. L., Christophers E. Lipopolysaccharide-stimulated human monocytes secrete, apart from neutrophil-activating peptide 1/interleukin 8, a second neutrophil-activating protein. NH2-terminal amino acid sequence identity with melanoma growth stimulatory activity. J Exp Med. 1990 Apr 1;171(4):1091–1100. doi: 10.1084/jem.171.4.1091. [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. Tekamp-Olson P., Gallegos C., Bauer D., McClain J., Sherry B., Fabre M., van Deventer S., Cerami A. Cloning and characterization of cDNAs for murine macrophage inflammatory protein 2 and its human homologues. J Exp Med. 1990 Sep 1;172(3):911–919. doi: 10.1084/jem.172.3.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Van Damme J., Van Beeumen J., Opdenakker G., Billiau A. A novel, NH2-terminal sequence-characterized human monokine possessing neutrophil chemotactic, skin-reactive, and granulocytosis-promoting activity. J Exp Med. 1988 Apr 1;167(4):1364–1376. doi: 10.1084/jem.167.4.1364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Walz A., Burgener R., Car B., Baggiolini M., Kunkel S. L., Strieter R. M. Structure and neutrophil-activating properties of a novel inflammatory peptide (ENA-78) with homology to interleukin 8. J Exp Med. 1991 Dec 1;174(6):1355–1362. doi: 10.1084/jem.174.6.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Walz A., Peveri P., Aschauer H., Baggiolini M. Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes. Biochem Biophys Res Commun. 1987 Dec 16;149(2):755–761. doi: 10.1016/0006-291x(87)90432-3. [DOI] [PubMed] [Google Scholar]
  26. Watanabe K., Iida M., Takaishi K., Suzuki T., Hamada Y., Iizuka Y., Tsurufuji S. Chemoattractants for neutrophils in lipopolysaccharide-induced inflammatory exudate from rats are not interleukin-8 counterparts but gro-gene-product/melanoma-growth-stimulating-activity-related factors. Eur J Biochem. 1993 May 15;214(1):267–270. doi: 10.1111/j.1432-1033.1993.tb17920.x. [DOI] [PubMed] [Google Scholar]
  27. Watanabe K., Kinoshita S., Nakagawa H. Purification and characterization of cytokine-induced neutrophil chemoattractant produced by epithelioid cell line of normal rat kidney (NRK-52E cell). Biochem Biophys Res Commun. 1989 Jun 30;161(3):1093–1099. doi: 10.1016/0006-291x(89)91355-7. [DOI] [PubMed] [Google Scholar]
  28. Watanabe K., Kinoshita S., Nakagawa H. Very rapid assay of polymorphonuclear leukocyte chemotaxis in vitro. J Pharmacol Methods. 1989 Aug;22(1):13–18. doi: 10.1016/0160-5402(89)90046-6. [DOI] [PubMed] [Google Scholar]
  29. Watanabe K., Koizumi F., Kurashige Y., Tsurufuji S., Nakagawa H. Rat CINC, a member of the interleukin-8 family, is a neutrophil-specific chemoattractant in vivo. Exp Mol Pathol. 1991 Aug;55(1):30–37. doi: 10.1016/0014-4800(91)90016-q. [DOI] [PubMed] [Google Scholar]
  30. Watanabe K., Konishi K., Fujioka M., Kinoshita S., Nakagawa H. The neutrophil chemoattractant produced by the rat kidney epithelioid cell line NRK-52E is a protein related to the KC/gro protein. J Biol Chem. 1989 Nov 25;264(33):19559–19563. [PubMed] [Google Scholar]
  31. Watanabe K., Suematsu M., Iida M., Takaishi K., Iizuka Y., Suzuki H., Suzuki M., Tsuchiya M., Tsurufuji S. Effect of rat CINC/gro, a member of the interleukin-8 family, on leukocytes in microcirculation of the rat mesentery. Exp Mol Pathol. 1992 Feb;56(1):60–69. doi: 10.1016/0014-4800(92)90023-5. [DOI] [PubMed] [Google Scholar]
  32. Yoshimura T., Matsushima K., Tanaka S., Robinson E. A., Appella E., Oppenheim J. J., Leonard E. J. Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9233–9237. doi: 10.1073/pnas.84.24.9233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yoshimura T., Yuhki N. Neutrophil attractant/activation protein-1 and monocyte chemoattractant protein-1 in rabbit. cDNA cloning and their expression in spleen cells. J Immunol. 1991 May 15;146(10):3483–3488. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES