Human B lymphoblastoid cell lines facilitate the growth in vitro of human NK cells and of T cell clones (1-4), and together with a source of IL-2, have been successfully used to maintain both NK and T cell clones in culture (1, 2). We have shown that irradiated B lymphoblastoid cell lines induce proliferation of purified human NK cells only in synergy with IL-2 (3). They also facilitate continued proliferation and enhance the cloning efficiency of purified human NK cells in limiting dilution assays in the presence of IL-2 without increasing the proportion (>507o) of NK cells entering the cell cycle in response to IL-2 (4). During culture of total PBMC with irradiated B cell lines, NK cells become activated, as shown by increased cytotoxic activity, by proliferation, and by expression of surface activation antigens such as class II HLA antigens, transferrin receptors, and IL-2 receptors (5, 6). In these cultures, a preferential proliferation of CD16+ CD56(NKH-1)+ CD3 -NK cells is observed (6) : in 10-d cultures, NK cell number is increased 25-fold, whereas T cell number is increased only 3-fold . Elimination of CD4 + cells or the presence of an anti-IL-2 antiserum completely prevents NK cell proliferation (6), suggesting that this probably depends on the production of IL-2 by CD4 + T cells upon allogeneic stimulation . However, the B cell lines also contribute directly to the proliferation of NK cells because in the absence of B cell lines neither high doses of IL-2 alone nor stimulation by allogeneic PBMC induce preferential proliferation of NK cells (6) .The mechanism by which B lymphoblastoid cell lines affect lymphocyte proliferation is not known . Studies with both human and murine lymphocytes (7, 8) suggest a role for immune interferon (IFN -'Y) in NK and T cell proliferation, although other studies (4) have shown that IFN-y production is not required. IFN-'Y is produced in cultures of thymocytes with irradiated B lymphoblastoid cell lines (9) . Low density murine spleen B cells (10) and certain human B cell lines (Cassatella, M . A .,
SummaryWe previously reported that natural killer cell stimulatory factor (NKSF), a heterodimeric lymphokine purified from the conditioned medium of human B lymphoblastoid cell lines, induces interferon y (IFN-y) production from resting peripheral blood lymphocytes (PBL) and synergizes with interleukin 2 in this activity. In this study, we show that human NKSF induces IFN-y production from both resting and activated human PBL and from freshly isolated murine splenocytes . Human T and NK cells produce IFN-,y in response to NKSF, but resting PBL require the presence of nonadherent human histocompatibility leukocyte antigens DR' (HLA DR+) accessory cells to respond to NKSF. The mechanism(s) by which NKSF induces IFN-y production results in accumulation of IFN-y mRNA, is insensitive to cyclosporin A, and synergizes with those mediated by phytohemmagglutinin, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens, but not by Ca 2+ ionophores. The ability of NKSF to directly induce IFN-,y production and to synergize with other physiological IFN-,y inducers, joined with the previously described ability to enhance lymphocyte cytotoxicity and proliferation, indicates that this lymphokine is a powerful immunopotentiating agent .
A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum-opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced Nformylmethionylleucylphenylalanine(FMLP)-stimulated degranulation ofCytochalasin B pretreated neutrophils and FMLPstimulated superoxide production. In contrast, rH GM-CSF did not promote adherence of granulocytes to endothelial cells or plastic surfaces. rH GM-CSF selectively enhanced the surface expression of granulocyte functional antigens 1 and 2, and the Mol antigen. rH GM-CSF induced morphological changes and enhanced the survival of both neutrophils and eosinophils by 6 and 9 h, respectively. These experiments show that granulocytemacrophage colony stimulating factor can selectively stimulate mature granulocyte function.
The present study shows that recombinant interleukin 2 (IL-2) purified to homogeneity induces a rapid and potent enhancement of spontaneous cytotoxicity of human peripheral blood lymphocytes. The cells mediating cytotoxicity after 18-h treatment with IL-2 have surface markers of natural killer (NK) cells and are generated from the peripheral blood subset containing spontaneous cytotoxic cells. A parallel production of gamma interferon (IFN-gamma) is induced by recombinant IL-2 (rIL-2), and NK cells appear to be the major producer cells, whereas T cells are unable to produce IFN-gamma under these experimental conditions. However, the kinetics of the enhancement of cytotoxicity are faster than those of IFN-gamma production, and monoclonal anti-IFN-gamma antibodies do not suppress this effect, making it unlikely that the IFN-gamma produced is responsible for the enhancement. The enhancement of NK cell activity induced by rIL-2 precedes any proliferative response of the lymphocytes, which is instead observed in longer-term cultures of both NK and T cells.
Interleukin-6 (IL-6) shares several biologic properties with IL-1, including hematopoietin-1 activity and stimulation of T cells. Because many of their biologic activities overlap, we developed and used a specific radioimmunoassay (RIA) for IL-6 to compare production of this cytokine on a molar basis with that of IL-1 alpha, IL-1 beta, and tumor necrosis factor (TNF)alpha. The RIA correlated well with the hybridoma bioassay for IL-6 (r = .87, P less than .001). Freshly isolated human peripheral blood mononuclear cells (PBMC) cultured in the absence of stimuli did not produce IL-6 in most cases. Kinetics of secretion and cell-association of IL-6 were studied. In contrast to IL-1 alpha but similar to TNF, IL-6 was almost entirely secreted into the extracellular fluid. Incubation with different stimuli (lipopolysaccharide [LPS], phytohemagglutinin [PHA], Staphylococcus epidermidis, or IL-1 alpha) resulted in production of IL-6. However, on a molar basis PBMC produced approximately two to three times less IL-6 than IL-1 alpha, IL-1 beta, or TNF, regardless of the stimulus. The amount of IL-6 produced from PBMC was consistent when measured in the same subjects six time during a 12-week period. In a cohort of 38 donors, the coefficient of variation for IL-6 production was .32, compared with .92 for IL-1 beta and .96 for TNF. Comparing cytokine production by PBMC, there was a significant correlation between IL-6 and IL-1 beta (r = .72) and between IL-6 and TNF (r = .66). IL-6 did not stimulate IL-1 beta or TNF production, but suppressed IL-1 beta and TNF production induced by LPS or PHA by 30% (P less than .01). This suppression of IL-1 beta and TNF by IL-6 appears to be on the level of transcription.
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