RAPID IL-2-INDUCED ADHERENCE OF HUMAN NATURAL-KILLER-CELLS - EXPRESSION OF MESSENGER-RNA FOR CYTOKINES AND IL-2 RECEPTORS IN ADHERENT NK-CELLS

Natural killer (NK) cells selected by IL-2-induced rapid adherence to plastic and called A-NK cells represent a phenotypically and functiona lly distinct subset of mature peripheral blood NK cells. To further ch aracterize this subset of NK cells functionally, their potential to ex press mRNA for the IL-2R and various cytokines after IL-2 activation w as examined. Highly purified normal human peripheral blood resting NK (R-NK) cells were obtained by negative immunoselection using OKT3 mAb and magnetic beads coated with goat anti-mouse Ig. By two-color flow c ytometry, >90% of these R-NK cells were either CD3-CD56+CD16+ or - or CD3-CD56-CD16+. R-NK cells were activated in the presence of 6000 IU/m l (22 nM) of IL-2 for different periods of time. After 1, 3, 5, or 24 h, plastic-adherent (A) and nonadherent (NA) NK cells were separated a nd compared for the expression of the IL-2R or cytokine mRNA by in sit u hybridization, using [S-35]-cDNA probes. Only low proportions of R-N K cells expressed genes for IL-2Rp55 (16%) or cytokines IL-2 (20%), IF N-gamma (18%), TNF-alpha (16%), and TGF-beta (7%). Thus, the genes for the IL-2Rp55 and these cytokines were not constitutively expressed by most human R-NK cells, and there was no indication that the NK cells used in these experiments were activated in vivo or during the purific ation procedure. However, larger proportions of R-NK cells showed expr ession of mRNA for IL-1-beta (35%) and IL-6 (40%), which indicates tha t genes for these cytokines may be constitutively expressed in a subst antial proportion of normal human circulating NK cells. When R-NK cell s were incubated in the presence of 22 nM of IL-2 for 1 to 24 h and se parated into A-NK cells and NA-NK cells, a large proportion of A-NK ce lls became positive for IL-2R and cytokine gene expression. In contras t, the proportion of mRNA-positive NA-NK cells was similar or lower th an that observed for R-NK cells, with the exception of an increase in TGF-beta. Thus, significantly higher proportions of A-NK than NA-NK ce lls expressed mRNA for the IL-2Rp75 (e.g, at 3 h after induction with IL-2, 58% vs 12%), IL-2Rp55 (at 5 h, 74% vs 4%), IL-2 (at 5 h, 68% vs 10%), TGF-beta (at 5 h, 82% vs 30%), IL-1-beta (at 5 h, 80% vs 24%) an d IL-6 (at 5 h, 79% vs 34%). Message for TNF-alpha or IFN-gamma was ex pressed in a high proportion of both A-NK and NA-NK cells (at 3 h, 70% and 64% for TNF-alpha and 58% and 30% for IFN-gamma, respectively). T he increased mRNA expression in A-NK cells at 3 h was followed by high levels of release of the proteins in 48 h culture supernatants. As ex pected, A-NK cell supernatants contained considerably higher levels of sIL-2R, IL-2, and IL-1-beta than those of NA-NK cells. In contrast, N A-NK cell supernatants contained more IFN-gamma than those of A-NK cel ls. Both subsets of NK cells produced similar levels of TNF-alpha. The data indicate that A-NK cells are a subset of NK cells that have not only been rapidly altered in their adherence properties, but also have a high level of NK activity and are selectively activated for express ion of IL-2R and several cytokine genes. Thus, A-NK cells appear to re present both a rapidly adherent and activated subset of NK cells.