Eur. J. Immunol. 1993. 23: 1831-1838 Regulation of cytokine production and soluble TNFR release in NK cells 1831 z Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin-2, interleukin-7 and interleukin-l2* Bjern Naume", Ann-Charlotte Johnsenv, Teje Espevik and Anders Sundan Institute of Cancer Research, University Medical Center, University of Trondheim, Trondheim Interleukin (1L)-2 IL-7 and IL-12 stimulate the generation of lymphokine- activated killer activity and proliferation in natural killer (NK) cells by different mechanisms. In this study,we have compared the ability of IL-2, IL-7 and IL-12 to induce expression of cytokines and cytokine receptors both at the gene and protein level. IL-2 and IL-12 stimulated the CD56+ NK cells to release significant amounts of soluble p55 and p75 tumor necrosis factor receptor (TNFR), whereas less amounts of solubleTNFR were detected in IL-7-stimulated cultures.The p55 and p75 TNFR mRNA were expressed in resting NK cells, and no further induction was observed after cytokine-stimulation. Compared to the effects of IL-2, IL-7 induced lower, but substantial levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA, and IL-7 was a more potent GM-CSF-inducing stimulus than IL-12. IL-12 induced higher levels of interferon- z y (IFN-y) mRNA than did IL-2, and IL-7 only weakly influenced the IF"-y expression. In accordance with the mRNA studies, IL-7 induced the secretion of high amounts of GM-CSF and no or low levels of IFN-y, whereas high amounts of IFN-y and low levels of GM-CSF were detected in supernatants from IL- 12-stimulated NK cells. In conclusion, IL-2, IL-7 and IL-12 differentially regulate expression of cytokines and cytokine receptors both at the gene and protein level. 1 Introduction Resting NK cells are highly responsive to IL-2, IL-7 and IL-12, -which all generate high lymphokine-activated killer (LAK) activity in these cells [l-61. NK cells are also able to synthezise a number of cytokines [7-111, involved in the modulation of hematopoiesis, immune responses and in the regulation of their own activities [4, 12-18]. Endogenously produced TNF-a is involved in the induction of NK cell activities by IL-2, IL-7 and IL-12 [4, 181. In accordance with this finding, these cytokines up-regulate cell surface p75 TNFR expression, but only low or no p55 TNFR expression is registered [3,4, 181. Recently, soluble extracellular parts of the twoTNFR were detected both in urine, serum and amniotic fluid in humans, and also in various tumor cell line cultures zyxwvutsr in vim [19-221. Further- [I 115161 * This work was supported by the Norwegian Cancer Society, Dynal A.S. (Norway), Nycomed BioReg AS (Norway) and The Norwegian Research Council for Science and Humanities (NAVF) . The contributions of the two first authors to this work should be considered as equal. Correspondence: Bjarn Naume, Institute of Cancer Research, University of Trondheim, Medisinsk Teknisk Senter, N-7005 Trondheim, Norway (Fax: 47 7 598801) Abbreviations: zyxwvutsr p55 TNFR: 55-kDa TNF receptor p75-TNFR: 75 kDa TNF receptor RT-PCR: Reverse tran- scriptase-polymerase chain reaction Key words: CD56+ NK cells/Interleukin-7/Interleukin-l2/Soluble tumor necrosis factor receptor more, activated granulocytes secrete soluble TNFR [23]. The release of TNFR may be an important regulatory factor of TNF-activities, as these receptor fragments both enhance and inhibit TNF effects in vitro at low and high TNFR levels, respectively [21,24]. Induction of soluble TNFR release in NK cells has so far not been reported, and a study of solubleTNFR in NK cells could bring new insight into the functional role of TNF in NK physiology. Some of the cytokines secreted by NK cells, for instance IFN-y and granulocyte-macrophage (GM)-CSF, have oppo- site effects on hematopoiesis and some immune responses [13-161. As previous reports indicate that IL-7 and IL-12 may activate NK cells differently [25], it is of interest to study possible differences in the cytokine expression pat- tern induced by these cytokines. This could reveal possible separate roles for IL-7 and IL-12 in immune responses involving NK cells. A substantial secretion of IFN-y has been reported in NK cells stimulated with IL-12 [26]. In another study, only low levels of IFN-y were detected in response to IL-7 [6]. The effects of IL-7 and IL-12 on the production of GM-CSF, as well as other cytokines (except IFN-y/TNF), have not been reported in NK cells. To adress these questions, the ability of IL-12, IL-7 and IL-2 to influence the expression of TNFR and various cytokines in highly purified NK cells was examined. For an initial screening regarding the expression of a number of cytokines and cytokine receptors we here have applied the reverse transcriptase-polymerase chain reaction (RT-PCR) with cytokine and cytokine receptor-specific primers. Although it is difficult to monitor changes in mRNA quantitatively with this technique, we reasoned that major changes would be realtively easy to detect under proper conditions. Such changes could then be quantitatively monitored at the protein level. zyxw 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1993 0014-2980/93/0808- 183 1$10.00 + .25/0