Production of granulocyte/macrophage-colony-stimulating factor by human natural killer cells. Modulation by the p75 subunit of the interleukin 2 receptor and by the CD2 receptor.
Resting natural killer (NK) cells express the p75 chain of the IL-2 receptor (IL-2R beta) and most NK cells express the CD2 (erythrocyte rosette) receptor. The cell adhesion molecule, LFA-3, is a natural co-ligand for CD2. Tac antigen (IL-2R alpha), a p55 IL-2R subunit, can be expressed after NK activation and may play a role in IL-2-induced NK proliferation. Little is known of the molecular mechanisms underlying cytokine production in NK cells. We investigated the roles of IL-2R alpha, IL-2R beta, and CD2/LFA-3 in the molecular regulation of NK cell granulocyte/macrophage-colony-stimulating factor (GM-CSF) production. Enriched populations of peripheral blood NK cells were separated into CD16-positive and CD16-negative fractions by flow cytometry; positively selected cells were greater than 97% positive for CD16 (the FcIII receptor for IgG which is present on almost all NK cells), less than 1% positive for the T cell antigen CD3, and did not demonstrate rearrangement of the T cell receptor beta chain gene by Southern blot. NK cell supernatants were harvested after 3-4 d of incubation with 0-100 U/ml IL-2, or after incubation with anti-CD2 (T11(3] MAb and sheep red blood cells (SRBC are a homologue for LFA-3). Parallel cell aliquots were harvested at 3-16 h for transcriptional run-on assays, S1 nuclease assays, and actinomycin D mRNA t1/2 determinations. IL-2-activated NK supernatants contained large amounts of GM-CSF (178 +/- 35 pg/ml) by ELISA as did supernatants from CD2-activated NK cells (T11(3) MAb + SRBC: 212 +/- 42) vs. less than 20 pg/ml for NK cells incubated alone or with either SRBC or T11(3) MAb alone. Sepharose-linked anti-CD3 MAb did not induce GM-CSF release from NK cells. By S1 analysis, both IL-2 and CD2 stimulation markedly augmented GM-CSF mRNA expression but with very different latencies of onset. IL-2R beta MAb inhibited greater than 85% of GM-CSF release from IL-2-activated NK cells and markedly suppressed IL-2-induced GM-CSF mRNA expression, whereas IL-2R alpha MAb even at 2,000-fold molar excess of IL-2 had little effect (less than 10%) on either GM-CSF release or mRNA expression. Run-on assays showed that GM-CSF is constitutively transcribed in NK cells and that IL-2 and CD2-activated cells had a three- to fourfold increased rate of GM-CSF transcription compared to nonstimulated cells. The t1/2 of GM-CSF mRNA in IL-2-activated NK cells was identical to that of unstimulated NK cells (15 min), whereas GM-CSF mRNA t1/2 in CD2-activated NK cells was increased 2.5-fold. We conclude that GM-CSF production in NK cells is regulated by both the IL-2Rbeta and the CD2 receptor but not by IL-2Ralpha, that both transcriptional and posttranscriptional signals act together to modulate the level of GM-CSF mRNA in NK cells, and that the molecular mechanisms underlying NK cell GM-CSF production are dependent in part on differential surface receptor activation.
Department of Medicine, Stanford University Medical Center, California 94305, USA.
L J
LJ
Levitt
A
A
Nagler
F
F
Lee
J
J
Abrams
M
M
Shatsky
D
D
Thompson
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000502:physiology*; D000943:Antigens, Differentiation / Q000502:physiology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology*; D018801:CD2 Antigens; D004920:Erythropoiesis; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D011961:Receptors, Fc / Q000502:physiology; D017452:Receptors, IgG; D011971:Receptors, Immunologic / Q000502:physiology*; D015375:Receptors, Interleukin-2 / Q000502:physiology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D007376:Interleukin-2; D012333:RNA, Messenger; D011961:Receptors, Fc; D017452:Receptors, IgG; D011971:Receptors, Immunologic; D015375:Receptors, Interleukin-2; D016178:Granulocyte-Macrophage Colony-Stimulating Factor