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. 1992 Nov 1;176(5):1387–1398. doi: 10.1084/jem.176.5.1387

Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells

PMCID: PMC2119437  PMID: 1357073

Abstract

Natural killer cell stimulatory factor (NKSF), or interleukin 12 (IL- 12), is a 70-kD heterodimeric cytokine composed of two covalently linked chains, p40 and p35. NKSF/IL-12 has multiple effects on T and NK cells and was originally identified and purified from the supernatant fluid of Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cell lines. We have produced a panel of monoclonal antibodies against both chains of NKSF/IL-12. Some of these antibodies have neutralizing activity, and several combinations of them have been used to establish sensitive radioimmunoassays detecting the free p40 chain, the free p35 chain, or the p70 heterodimer. Using these reagents, we have determined that most EBV-transformed human B lymphoblastoid cell lines constitutively produce low levels of the p70 heterodimer and an excess of the free p40 chain, whereas Burkitt lymphoma-derived, T, myeloid, and many solid tumor-derived cell lines produce neither. Production of both p40 and p70 is increased several-fold upon stimulation of the EBV- transformed cell lines with phorbol diesters. The ability of supernatant fluids from unstimulated and phorbol diester-stimulated cell lines to induce interferon gamma (IFN-gamma) production from T and NK cells, one of the effects of NKSF/IL-12, parallels the levels of production of the p70 heterodimer, known to be the biologically active form of NKSF/IL-12. Staphylococcus aureus Cowan I strain (SAC) and other stimuli induce accumulation of p40 mRNA and production of both p40 and p70 by peripheral blood mononuclear cells (PBMC). The producer cells appear to include both adherent cells and nonadherent lymphocytes, possibly B cells. The supernatant fluids from SAC- stimulated PBMC mediate the typical functions of NKSF/IL-12 (i.e., IFN- gamma induction, mitogenic effects on T/NK blasts, enhancement of NK cell cytotoxicity) at concentrations of p70 similar to those at which recombinant NKSF/IL-12 mediates the same functions. Moreover, these activities are significantly inhibited by anti-NKSF/IL-12 antibodies. The neutralizing anti-NKSF/IL-12 antibodies also inhibit 85% of the IFN- gamma production in response to SAC, an NKSF/IL-12 inducer, and approximately 50% of the IFN-gamma production in response to non- NKSF/IL-12-inducers such as IL-2, phytohemagglutinin, and anti-CD3 antibodies. These results indicate that induced or constitutively produced NKSF/IL-12 has a major role in facilitating IFN-gamma production by peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

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Selected References

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  1. Bancroft G. J., Sheehan K. C., Schreiber R. D., Unanue E. R. Tumor necrosis factor is involved in the T cell-independent pathway of macrophage activation in scid mice. J Immunol. 1989 Jul 1;143(1):127–130. [PubMed] [Google Scholar]
  2. Cassatella M. A., Bazzoni F., Calzetti F., Guasparri I., Rossi F., Trinchieri G. Interferon-gamma transcriptionally modulates the expression of the genes for the high affinity IgG-Fc receptor and the 47-kDa cytosolic component of NADPH oxidase in human polymorphonuclear leukocytes. J Biol Chem. 1991 Nov 25;266(33):22079–22082. [PubMed] [Google Scholar]
  3. Cassatella M. A., Hartman L., Perussia B., Trinchieri G. Tumor necrosis factor and immune interferon synergistically induce cytochrome b-245 heavy-chain gene expression and nicotinamide-adenine dinucleotide phosphate hydrogenase oxidase in human leukemic myeloid cells. J Clin Invest. 1989 May;83(5):1570–1579. doi: 10.1172/JCI114054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chehimi J., Starr S. E., Frank I., Rengaraju M., Jackson S. J., Llanes C., Kobayashi M., Perussia B., Young D., Nickbarg E. Natural killer (NK) cell stimulatory factor increases the cytotoxic activity of NK cells from both healthy donors and human immunodeficiency virus-infected patients. J Exp Med. 1992 Mar 1;175(3):789–796. doi: 10.1084/jem.175.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chizzonite R., Truitt T., Podlaski F. J., Wolitzky A. G., Quinn P. M., Nunes P., Stern A. S., Gately M. K. IL-12: monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts. J Immunol. 1991 Sep 1;147(5):1548–1556. [PubMed] [Google Scholar]
  6. Devalon M. L., Elliott G. R., Regelmann W. E. Oxidative response of human neutrophils, monocytes, and alveolar macrophages induced by unopsonized surface-adherent Staphylococcus aureus. Infect Immun. 1987 Oct;55(10):2398–2403. doi: 10.1128/iai.55.10.2398-2403.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gately M. K., Desai B. B., Wolitzky A. G., Quinn P. M., Dwyer C. M., Podlaski F. J., Familletti P. C., Sinigaglia F., Chizonnite R., Gubler U. Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor). J Immunol. 1991 Aug 1;147(3):874–882. [PubMed] [Google Scholar]
  8. Gubler U., Chua A. O., Schoenhaut D. S., Dwyer C. M., McComas W., Motyka R., Nabavi N., Wolitzky A. G., Quinn P. M., Familletti P. C. Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4143–4147. doi: 10.1073/pnas.88.10.4143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kaufman R. J. Selection and coamplification of heterologous genes in mammalian cells. Methods Enzymol. 1990;185:537–566. doi: 10.1016/0076-6879(90)85044-o. [DOI] [PubMed] [Google Scholar]
  10. Kobayashi M., Fitz L., Ryan M., Hewick R. M., Clark S. C., Chan S., Loudon R., Sherman F., Perussia B., Trinchieri G. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med. 1989 Sep 1;170(3):827–845. doi: 10.1084/jem.170.3.827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Murphy M., Loudon R., Kobayashi M., Trinchieri G. Gamma interferon and lymphotoxin, released by activated T cells, synergize to inhibit granulocyte/monocyte colony formation. J Exp Med. 1986 Jul 1;164(1):263–279. doi: 10.1084/jem.164.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Perussia B., Ramoni C., Anegon I., Cuturi M. C., Faust J., Trinchieri G. Preferential proliferation of natural killer cells among peripheral blood mononuclear cells cocultured with B lymphoblastoid cell lines. Nat Immun Cell Growth Regul. 1987;6(4):171–188. [PubMed] [Google Scholar]
  14. Perussia B., Starr S., Abraham S., Fanning V., Trinchieri G. Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. I. Characterization of the lymphocyte subset reactive with B73.1. J Immunol. 1983 May;130(5):2133–2141. [PubMed] [Google Scholar]
  15. Scott P. IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis. J Immunol. 1991 Nov 1;147(9):3149–3155. [PubMed] [Google Scholar]
  16. Shokri F., Mageed R. A., Maziak B. R., Jefferis R. Expression of VHIII-associated cross-reactive idiotype on human B lymphocytes. Association with staphylococcal protein A binding and Staphylococcus aureus Cowan I stimulation. J Immunol. 1991 Feb 1;146(3):936–940. [PubMed] [Google Scholar]
  17. Stern A. S., Podlaski F. J., Hulmes J. D., Pan Y. C., Quinn P. M., Wolitzky A. G., Familletti P. C., Stremlo D. L., Truitt T., Chizzonite R. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6808–6812. doi: 10.1073/pnas.87.17.6808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wherry J. C., Schreiber R. D., Unanue E. R. Regulation of gamma interferon production by natural killer cells in scid mice: roles of tumor necrosis factor and bacterial stimuli. Infect Immun. 1991 May;59(5):1709–1715. doi: 10.1128/iai.59.5.1709-1715.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wolf S. F., Temple P. A., Kobayashi M., Young D., Dicig M., Lowe L., Dzialo R., Fitz L., Ferenz C., Hewick R. M. Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells. J Immunol. 1991 May 1;146(9):3074–3081. [PubMed] [Google Scholar]

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