Cloning, expression, biologic properties, and transcription during endotoxemia

Joseph Gerard Cannon, B. D. Clark, P. Wingfield, U. Schmeissner, C. Losberger, C. A. Dinarello, A. R. Shaw

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Abstract

The cloning, sequencing, expression, and biologic activities of rabbit IL-1 α and β are described. A cDNA library was constructed in λgt10 by using polyadenylated RNA extracted from rabbit adherent splenic macrophages 4 h after stimulation with endotoxin. By using the cDNA for human IL-1β and IL-1α as hybridization probes, cDNA for both forms of rabbit IL-1 were isolated. The cDNA for rabbit IL-1β encodes a precursor polypeptide of 268 amino acids with an overall homology to human IL-1β of 74% (81% in the mature region coding for a 17.5 kDa carboxyl-terminal protein). The similarity between the two rabbit IL-1 forms is 31% for the entire molecule and 34% for the mature protein. The mature polypeptides of both forms were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity and tested in a variety of biologic assays. Both forms produced typical endogenous pyrogen fevers in rabbits and augmented murine thymocyte and Th cell proliferation. Rabbit IL-1 α and β were more pyrogenic in rabbits than human rIL-1β, whereas human rIL-1 α and β were slightly more potent lymphocyte-activating factors. The recombinant rabbit proteins induced PGE and IL-1 production from human PBMC in vitro. A RIA for human IL-1α did not recognize rabbit IL-1 α or β, but rabbit IL-1β cross-reacted (as much as 30%) in a RIA for human IL-1β. Rabbits were injected with endotoxin and mRNA for both forms of IL-1 were observed primarily in the spleen and liver. The mRNA reached maximal levels after 60 min, then declined rapidly over the next 3 h, but were still present after 24 h. Liver tissue removed 4 h after endotoxin infusion produced lymphocyte-activating factors which were neutralized by more than 90% with a combination of goat anti-rabbit IL-1α and anti-IL-1β.

Original languageEnglish (US)
Pages (from-to)2299-2306
Number of pages8
JournalJournal of Immunology
Volume142
Issue number7
StatePublished - Jan 1 1989

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Endotoxemia
Interleukin-1
Organism Cloning
Rabbits
Endotoxins
Complementary DNA
Recombinant Proteins
Messenger RNA
Peptides
Liver

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Cannon, J. G., Clark, B. D., Wingfield, P., Schmeissner, U., Losberger, C., Dinarello, C. A., & Shaw, A. R. (1989). Cloning, expression, biologic properties, and transcription during endotoxemia. Journal of Immunology, 142(7), 2299-2306.

Cloning, expression, biologic properties, and transcription during endotoxemia. / Cannon, Joseph Gerard; Clark, B. D.; Wingfield, P.; Schmeissner, U.; Losberger, C.; Dinarello, C. A.; Shaw, A. R.

In: Journal of Immunology, Vol. 142, No. 7, 01.01.1989, p. 2299-2306.

Research output: Contribution to journalArticle

Cannon, JG, Clark, BD, Wingfield, P, Schmeissner, U, Losberger, C, Dinarello, CA & Shaw, AR 1989, 'Cloning, expression, biologic properties, and transcription during endotoxemia', Journal of Immunology, vol. 142, no. 7, pp. 2299-2306.
Cannon JG, Clark BD, Wingfield P, Schmeissner U, Losberger C, Dinarello CA et al. Cloning, expression, biologic properties, and transcription during endotoxemia. Journal of Immunology. 1989 Jan 1;142(7):2299-2306.
Cannon, Joseph Gerard ; Clark, B. D. ; Wingfield, P. ; Schmeissner, U. ; Losberger, C. ; Dinarello, C. A. ; Shaw, A. R. / Cloning, expression, biologic properties, and transcription during endotoxemia. In: Journal of Immunology. 1989 ; Vol. 142, No. 7. pp. 2299-2306.
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abstract = "The cloning, sequencing, expression, and biologic activities of rabbit IL-1 α and β are described. A cDNA library was constructed in λgt10 by using polyadenylated RNA extracted from rabbit adherent splenic macrophages 4 h after stimulation with endotoxin. By using the cDNA for human IL-1β and IL-1α as hybridization probes, cDNA for both forms of rabbit IL-1 were isolated. The cDNA for rabbit IL-1β encodes a precursor polypeptide of 268 amino acids with an overall homology to human IL-1β of 74{\%} (81{\%} in the mature region coding for a 17.5 kDa carboxyl-terminal protein). The similarity between the two rabbit IL-1 forms is 31{\%} for the entire molecule and 34{\%} for the mature protein. The mature polypeptides of both forms were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity and tested in a variety of biologic assays. Both forms produced typical endogenous pyrogen fevers in rabbits and augmented murine thymocyte and Th cell proliferation. Rabbit IL-1 α and β were more pyrogenic in rabbits than human rIL-1β, whereas human rIL-1 α and β were slightly more potent lymphocyte-activating factors. The recombinant rabbit proteins induced PGE and IL-1 production from human PBMC in vitro. A RIA for human IL-1α did not recognize rabbit IL-1 α or β, but rabbit IL-1β cross-reacted (as much as 30{\%}) in a RIA for human IL-1β. Rabbits were injected with endotoxin and mRNA for both forms of IL-1 were observed primarily in the spleen and liver. The mRNA reached maximal levels after 60 min, then declined rapidly over the next 3 h, but were still present after 24 h. Liver tissue removed 4 h after endotoxin infusion produced lymphocyte-activating factors which were neutralized by more than 90{\%} with a combination of goat anti-rabbit IL-1α and anti-IL-1β.",
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