Activation of human natural killer cells by lipopolysaccharide and generation of interleukin-1 alpha, beta, tumour necrosis factor and interleukin-6. Effect of IL-1 receptor antagonist.

The presence in the body of an antigen species or a bacterial lipopolysaccharide (LPS) has a pleiotropic effect on the immune system activating macrophages, lymphocytes and natural killer (NK) cells. Recently it has been reported that human macrophages not only secrete interleukin-1 (IL-1) but also its inhibitor, called IL-1 receptor antagonist (IL-1ra), structurally similar to IL-1 beta, but with no IL-1-like activity and which binds to the IL-1 receptor. In this study we show that LPS stimulates NK cell activity and IL-1ra potentiates the stimulatory effect of human recombinant interleukin-2 (hrIL-2) on NK cell activity. In addition, we found that hrIL-1ra inhibits DNA synthesis in lymphocyte culture stimulated with phytohaemagglutinin (PHA) (20 micrograms/ml), presumably via IL-1 inhibition. We also found that LPS is a potent stimulator of monokines: IL-6, tumour necrosis factor-alpha (TNF-alpha), and IL-1 beta, as determined by radioimmunoassay method, and interferon-gamma (IFN-gamma), IL-2, TNF-alpha and IL-1 alpha, as determined by ELISA method, in peripheral blood mononuclear cells (PBMC). We used PBMC as effector cells since LPS requires the presence of accessory cells to activate lymphocytes and bind to the HLA-DR molecule on accessory cells. The effect of LPS on PBMC cytotoxicity has been compared with an endotoxin-free extract of Escherichia coli, OM-8990, which did not provoke cytokine production nor did it cause enhancement of NK cell activity. We found that human recombinant IL-1ra potentiates the stimulatory effect of IL-2 on NK cell activity, similar to hrIL-1 beta. The potentiation of IL-2 in stimulating NK cell activity by IL-1ra is not yet understood. Since IL-1ra is a part of the IL-1 family, it may work in a similar fashion to IL-1, which also potentiates IL-2 to enhance NK cell activity but has been shown not to be directly important in tumour cell killing. In addition, hrIL-1ra can amplify the effect of IL-2 on NK activity, possibly by inhibiting the cyclo-oxygenase products, which are immunosuppressive and are generated in antigen-stimulated PBMC cultures. The generation of IFN-gamma by PBMC after treatment with LPS strongly suggests that the enhancement of NK cell activity may be indirectly due to IFN production.     

Restoration of anti-interleukin-1 depressed natural killer activity by human recombinant interferon alpha or gamma, human recombinant interleukin-2 and indomethacin

The lymphokines interleukin-2 and interferon alpha or gamma are synthesized and secreted by activated mononuclear cells (MC) and play a critical role in the proliferative expansion of T-lymphocyte effector cells during the immune response. The pretreatment of human peripheral blood mononuclear cells (PBMC) with antibody IgG against human interleukin-1 (IL-1) from normal rabbit serum, inhibited their natural killer (NK) activity against both myeloid (K562) and lymphoid (MOLT-4) cell lines. Percent specific lysis of tumor cells decreased by increasing the antibody anti-IL-1 dose in an almost linear fashion. When the effector cells were pretreated with human recombinant interleukin-2, human interferon alpha or gamma and indomethacin alone or in combination, the inhibitory effect of antibody against human IL-1 was almost totally reversed.     

Enhancing effect of natural killer cell stimulatory factor (NKSF/interleukin-12) on cell-mediated cytotoxicity against tumor-derived and virus-infected cells

Natural killer cell stimulatory factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine with pleiomorphic effects on T and NK cells, including induction of lymphokine production, mitogenesis, and enhancement of spontaneous cytotoxic activity. Similarly to IL-2, NKSF/IL-12 enhances NK cell-mediated cytotoxicity within a few hours and independently from induced proliferation. This effect is independent from other induced cytokines, because it is not prevented by antibodies neutralizing interferon (IFN)-α, IFN-β IFN-γ, IL-2 or tumor necrosis factor (TNF)-α and, unlike the induction of IFN-γ production by peripheral blood lymphocytes, it does not require HLA class II-positive accessory cells. Enhanced cytotoxicity is accompanied by morphologic changes in NK cells, including a significant increase in the number of cytoplasmic granules. In addition to the previously described ability to enhance the cytotoxic activity of NK cells against tumor-derived target cells, NKSF/IL-12 is also a potent stimulator of cytotoxicity against virus-infected cells, either fibroblasts acutely infected with herpes viruses or T cell lines chronically infected with human immunodeficiency virus-1. NK cell-mediated antibody-dependent cytotoxicity or anti-CD16 antibody-redirected lysis is not significantly enhanced by NKSF/IL-12. However, the ability of resting peripheral blood T cells to mediate anti-CD3 antibody-redirected lysis is enhanced by 18-h incubation with NKSF/IL-12, indicating that this lymphokine can modulate the cytotoxic capability of both NK and T cells.     

IL-10 stimulatory effects on human NK cells explored by gene profile analysis

The molecular mechanisms underlying the increase of natural killer (NK) cell anticancer activity mediated by interleukin (IL)-10 have not been elucidated. The aim of this study was to identify potential molecular mediators of IL-10 stimulatory effects by exploring the NK cell gene display induced by this cytokine. Gene profile was determined by high-throughput cDNA microarray and quantitative real-time PCR. In vitro, NK cells resting or conditioned with IL-10 were tested for cytotoxicity, migration and proliferation. IL-10 enhanced mRNA levels of cell activation/cytotoxicity-related genes (eg secretogranin, TIA-1, HMG-1, interferon-inducible genes) not upregulated by IL-2. In line with these findings, IL-10 increased NK cell in vitro cytotoxicity against Daudi cells. Unlike IL-2, IL-10 did not show any significant effect on NK cell in vitro proliferation and migration. However, gene profile analysis showed that IL-10 increased the expression of cell migration-related genes (eg L-selectin, vascular endothelium growth factor receptor-1, plasminogen activator, tissue; formyl peptide receptor, lipoxin A4 receptor), which might support a stimulatory effect not evident with the in vitro functional assay. Overall, gene profiling allowed us to formulate new hypotheses regarding the molecular pathways underlying the stimulatory effects of IL-10 on NK cells, supporting further investigation aimed at defining its role in cancer immune rejection.     

Depletion of endogenous interleukin-10 augments interleukin-1 beta secretion by Mycobacterium bovis BCG-reactive human cells.

In this study, we found evidence that the interleukin-10 (IL-10) protein is functionally relevant in Mycobacterium bovis BCG-induced cytokine synthesis, as neutralization of endogenously synthesized IL-10 in human cells activated with BCG resulted in a two- to threefold increase in the level of IL-1 beta. When exogenous recombinant human IL-10 was added to human mononuclear cells, a significant reduction of BCG-induced IL-1 beta secretion was observed. This inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in the presence of IL-10, and it was specific, as it was completely abolished in the presence of anti-IL-10 neutralizing monoclonal antibody while an irrelevant antibody used as a control had no effect. Taken together, these are the first studies that demonstrate that the depletion of endogenous IL-10 via anti-IL-10 antibody results in a very significantly enhanced BCG-induced IL-1 beta secretion and that the addition of exogenous IL-10 to human mononuclear cells stimulated with BCG inhibits IL-1 beta production. Further experimental work is needed to determine if the neutralization of IL-10 activity via anti-IL-10 antibody indeed enhances cytokine synthesis in vivo. However, the present results may be of importance, since the use of anti-IL-10 antibody could presumably contribute to the protective immunity induced by BCG against tuberculosis via an increase in cytokine synthesis that would amplify antimicrobial systems.     

Role of interleukin-18 in human natural killer cell is associated with interleukin-2

Human natural killer (NK) cells constitute an important cellular component of innate immunity, capable of killing infected and transformed cells. The proliferation and activation of NK cells are regulated by various cytokines. Interleukin-18 (IL-18) promotes NK cell activation; however, whether the effects of IL-18 on NK cell are associated with other cytokines is still unknown. In this study, we observed that IL-18 induced NK cell apoptosis and inhibited NK cell expansion in the presence of low concentrations of interleukin-2 (IL-2), while high concentrations of IL-2 overcame these effects of IL-18, and high concentrations of IL-2 promoted the stimulatory activity of IL-18 on NK cells. At a low concentration of IL-2, IL-18 induced NK cell apoptosis in part through activation of the FasL/Fas- and TNFα/TNFR-mediated death receptor signaling by enhancing FasL expression and inhibiting c-FLIP(long) expression. However, high concentrations of IL-2 strongly blocked IL-18-induced NK cell apoptosis through alleviating IL-18-induced FasL expression and activation of Fas-mediated death signaling and increasing anti-apoptosis molecule (Bcl-X(L)). These results reveal that the effects of IL-18 on human NK cell are associated with IL-2 concentration and suggest the importance of IL-2 level in cytokine immunotherapy.     

Cellulose acetate beads induce release of interleukin-1 receptor antagonist, but not tumour necrosis factor-α or interleukin-1β in human peripheral blood

OBJECTIVE: Dramatic improvements in clinical symptoms of rheumatoid arthritis and ulcerative colitis were observed after patients received granulocyte and monocyte adsorptive apheresis with a column containing cellulose acetate (CA) beads as adsorptive carriers. This study was to investigate the effect of CA beads on the generation of anti-inflammatory and pro-inflammatory cytokines in human blood. MATERIALS AND METHODS: We incubated human whole blood with CA beads at 37 degrees C for up to 2 h and measured tumour necrosis factor-alpha (TNF-alpha) interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra) produced by leucocytes. IL-1ra was also measured at the inflow and outflow of a column containing CA beads as leucocyte adsorptive carriers for the treatment of patients with ulcerative colitis. RESULTS: CA beads induced significant release of IL-1ra from leucocytes, but not TNF-alpha or IL-1beta. In contrast, all three cytokines were released when leucocytes were stimulated with lipopolysaccharide. IL-1ra was also markedly elevated in the outflow of the leucocyte apheresis column. CONCLUSIONS: These results indicate that CA beads selectively induce IL-1ra release from leucocytes which should contribute to the anti-inflammatory effect of granulocyte and monocyte adsorptive apheresis with CA beads as apheresis carriers.     

Reduced mitogen stimulation of DNA synthesis in human lymphocytes by a human recombinant interleukin-1 receptor antagonist.

The monokine interleukin-1 is produced by monocytes/macrophages after antigen/LPS stimulation and is an important early signal for the activation of resting T cells to become antigen specific T cells. However, little is known about the regulation and inhibition of IL-1. Recently, a new monokine has been described, generated by human macrophages, called interleukin-1 receptor antagonist (IL-1ra). This new monokine adheres to IL-1 in solution and blocks IL-1 receptor binding. IL-1ra is a glycoprotein structurally similar to IL-1 beta but having no interleukin-1-like activity. Using as a model mitogen (PHA 20 micrograms/ml)-stimulated lymphocyte DNA synthesis, we found that hrIL-1ra (30 min lymphocyte pretreatment) inhibits [3H]thymidine incorporation in a dose-dependent manner. This effect is most probably due to the inhibition of endogenous IL-1, which is a very important signal for T cell activation. The inhibition was maximum at the highest hrIL-1ra concentration used (250 ng/ml). However, when hrIL-1ra was added 2 h after PHA (20 micrograms/ml), a little, if any, inhibition of lymphocyte proliferation was found. The addition of hrIL-1ra simultaneously to the cell cultures with [3H]thymidine [( 3H]TdR) 6 h before the end of culture incubation did not significantly modify the results compared to the cells treated with PHA alone, indicating no interference of hrIL-1ra on [3H]TdR lymphocyte incorporation. We also found that the antibody anti-IL-1 beta inhibits mitogen stimulated lymphocyte DNA synthesis in dose-dependent concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of interleukin-18 in human natural killer cell is associated with interleukin-2

Human natural killer (NK) cells constitute an important cellular component of innate immunity, capable of killing infected and transformed cells. The proliferation and activation of NK cells are regulated by various cytokines. Interleukin-18 (IL-18) promotes NK cell activation; however, whether the effects of IL-18 on NK cell are associated with other cytokines is still unknown. In this study, we observed that IL-18 induced NK cell apoptosis and inhibited NK cell expansion in the presence of low concentrations of interleukin-2 (IL-2), while high concentrations of IL-2 overcame these effects of IL-18, and high concentrations of IL-2 promoted the stimulatory activity of IL-18 on NK cells. At a low concentration of IL-2, IL-18 induced NK cell apoptosis in part through activation of the FasL/Fas- and TNFα/TNFR-mediated death receptor signaling by enhancing FasL expression and inhibiting c-FLIP_long expression. However, high concentrations of IL-2 strongly blocked IL-18-induced NK cell apoptosis through alleviating IL-18-induced FasL expression and activation of Fas-mediated death signaling and increasing anti-apoptosis molecule (Bcl-X_L). These results reveal that the effects of IL-18 on human NK cell are associated with IL-2 concentration and suggest the importance of IL-2 level in cytokine immunotherapy.     

Comparison of the effects of interleukin-1α, interleukin-lβ and interferon-γ-inducing factor on the production of interferon-γ by natural killer

Interferon-γ inducing factor (IGIF) is a recently identified cytokine which stimulates the production of interferon-γ (IFN-γ) by T cells and enhances natural killer (NK) cell cytolytic activity. Protein fold recognition, structure prediction and comparative modeling have revealed that IGIF is a member of the interleukin (IL)-1 cytokine family and has prompted the designation IL-1γ. Here we report functional similarities between members of the IL-1 family by comparing the effects of IL-1α, IL-1β and IGIF on NK cell production of IFN-γ. All three IL-1 types enhanced NK cell production of IFN-γ when induced by IL-2 or IL-12, although at high concentrations (>10 ng/ml), IGIF was five- to tenfold more potent than IL-1α or IL-1β. This effect correlated with enhanced levels of mRNA for IFN-γ when NK cells were stimulated with IGIF plus IL-12. In contrast to IL-12 and IL-2, the ability of IGIF to stimulate NK cell production of IFN-γ was not increased by IL-1α or IL-1β. The ability of IGIF to enhance IFN-γ production was independent of the type I and type II IL-1 receptors or the IL-1R accessory protein. Together, these results identify IGIF as a potent stimulator of NK cell production of IFN-γ and demonstrate that the effect of IGIF on NK cell production of IFN-γ is similar to that of IL-1α and IL-1β but distinct from that of IL-12.     

Lipopolysaccharide induces human interleukin-1 receptor antagonist and interleukin-1 production in the same cell.

A new member of the interleukin-1 (IL-1) family has recently been described. Human IL-1 receptor antagonist (IL-1ra) is structurally related to IL-1 alpha and IL-1 beta but binds to IL-1 receptors on various target cells without demonstrable agonist activity. Understanding the mechanisms of regulation of IL-1ra production may clarify the biology of this unique cytokine as well as elucidate its possible role as a natural anti-inflammatory protein. The effects of lipopolysaccharide (LPS) on IL-1 alpha, IL-1 beta and IL-1ra production was studied at a single-cell level by use of cytokine-specific antibodies and indirect immunofluorescence technique. The peak synthesis of IL-1ra and IL-1 alpha/beta occurred in peripheral blood monocytes obtained from healthy blood donors within 4 and 6 h of cell stimulation, respectively. By double-staining procedure all IL-1ra-positive cells were also IL-1 alpha and/or beta positive. Thus, endotoxin induced simultaneous synthesis of the IL-1 gene family in the same cells. Only monocytes contributed to the production of IL-1 alpha, beta and IL-1ra during the 96 h of cell culture. The maximum number of IL-1ra-producing monocytes was 48 +/- 16% as compared to peak production of IL-1 alpha and beta which occurred in 75 +/- 9% and 80 +/- 12% (p < 0.001), respectively, of all peripheral blood monocytes. The incidence of IL-1 alpha- and beta-containing cells was not only significantly higher but also occurred for a longer time period, 72 h as compared to 24 h for IL-1ra localized in the Golgi organelle. However, IL-1ra-containing cells with a diffuse cytoplasmic appearance were also evident (20%-30%) at a later stage, 12 to 72 h after stimulation. Blocking IL-1 surface receptors by addition of exogenous recombinant IL-1 beta before stimulation could not inhibit the diffuse cytosolic localization. This indicates that the "late" staining pattern did not reflect IL-1ra being secreted and internalized after binding to extracellular receptors. Thus, perhaps IL-1ra modulates IL-1 effector mechanisms by receptor interactions both inside and outside the cell.     

Immunoglobulin D enhances the release of tumor necrosis factor-alpha, and interleukin-1 beta as well as interleukin-1 receptor antagonist from human mononuclear cells.

Immunoglobulin D (IgD) is normally present in only low concentrations in serum. In the hyper-IgD and periodic fever syndrome (HIDS), however, serum levels exceed 140 mg/l. This syndrome is further characterized by recurrent inflammatory febrile attacks together with an acute phase response and appearance of cytokines in the circulation. The role of IgD in the pathogenesis of HIDS and its relation to the increased cytokine concentrations is unclear. Therefore, we tested whether IgD, IgG and alpha 1-acid glycoprotein (AGP) isolated from human serum influence the synthesis of interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), and IL-1ra, as measured by specific radioimmunoassays, in human peripheral blood mononuclear cells (PBMC). Incubation of PBMC with IgD and AGP for 24 hr led to increased release of IL-1 beta, TNF-alpha, and IL-lra. The magnitude of stimulation of IgD exceeded that of AGP; the effect by IgD was dose-dependent and showed a 30-fold (TNF-alpha) to almost 150-fold (IL-1 beta) increase at the highest concentration (50 mg/l), while AGP (750 micrograms/ml) only increased the cytokine secretion fourfold (TNF-alpha) to almost 30-fold (IL-1 beta). The effect of IgD on IL-1ra was less dramatic but a fivefold increase was observed at 50 mg/l compared with a 2.5-fold increase with AGP. IgD potentiated the effect of lipopolysaccharide (LPS) on secretion of both IL-1 beta and TNF-alpha, although the effect was most apparent for TNF-alpha. Apart from inducing IL-1ra synthesis, IgG did not influence cytokine release in human PBMC. These data indicate that IgD is a potent inducer of TNF-alpha, IL-1 beta and IL-1ra and thus may contribute to the pathogenesis of HIDS.     

Mucosal cytokine therapy: marked antiviral and antitumor activity.

Mucosal administration of the Th1 stimulatory cytokines interleukin-2 (IL-2), IL-12, IL-15, IL-18, or granulocyte-macrophage colony-stimulating factor (GM-CSF) induced antiviral activity in mice challenged systemically with a lethal dose of encephalomyocarditis virus (EMCV) similar to that observed following parenteral administration. In contrast, mucosal administration of the Th2 stimulatory cytokines IL-4, IL-5, IL-10, or IL-13 did not affect significantly the survival of EMCV-infected animals. Mucosal administration of IL-2 or IL-12 also exerted a marked antitumor activity in mice inoculated intravenously with Friend erythroleukemia cells. Recombinant IL-2 and IL-18, but none of the other recombinant cytokines tested, induced low levels of IFN in vitro. Polyclonal antibodies to both mouse and human interferon-alpha/beta (IFN-alpha/beta) abrogated the antiviral activity of IL-2 in vivo, even though the anti-human IFN-alpha/beta antibody did not neutralize mouse IFN-alpha/beta, and neither antibody bound to IL-2. IL-15 did not exhibit antiviral activity in IFN-alpha/beta R-/- mice, which are deficient in natural killer (NK) cell activity. These results suggest that mucosal Th1 cytokine therapy induces a soluble factor or activates a specific cell population in the lymphoid or epithelial tissue of the oropharyngeal cavity, which potentiates elimination of virus-infected or neoplasic cells systemically.     

Enhancement of interleukin-1 activity by murine cytomegalovirus infection of a macrophage cell line

The effect of murine cytomegalovirus (MCMV) infection on interleukin 1 (IL-1) secretion was assessed using the macrophage cell lines P388D1 and J774A.1. The former proved to be nonpermissive for MCMV in that infectious virus and viral immediate early protein (pp89) were not expressed in these cells. MCMV infection of the P388D1 cells had no effect on release of biologically active IL-1. In contrast, J774A.1 cells, which were semipermissive for virus replication and pp89 expression, secreted enhanced levels of IL-1 activity following infection. The enhancement was evident when infection either preceded or followed lipopolysaccharide stimulation of the macrophages. The relative proportion of IL-1 alpha and beta secreted from MCMV-infected cells was similar to noninfected controls. In addition, the bioactivity of intracellular IL-1 alpha escaping membranes of fixed cells was unaffected by virus infection. From these findings, we conclude that limited MCMV expression in the J774A.1 macrophage cell line enhances secretion of IL-1 alpha and beta bioactivity.     

Reversal of human immunodeficiency virus type 1 protein-induced inhibition of natural killer cell activity by alpha interferon and interleukin-2

A recombinant fusion peptide, Env-Gag, derived from the human immunodeficiency virus type 1 (HIV-1) genome corresponding to a defined portion of the envelope (Env) and internal core (Gag) proteins was examined for immunoregulatory effects on the cytotoxic activity of natural killer (NK) cell-enriched, large granular lymphocytes (LGL) from healthy donors. Percoll-separated, NK cell-enriched LGL precultured for 24 h with Env-Gag at 10- and 50-ng/ml concentrations, which significantly stimulated lymphocyte proliferation, caused significant suppression of NK cell activity. Denatured Env-Gag did not cause any effect on the NK cell activity of LGL. Two other control peptides, one derived from the Escherichia coli vector used to clone the HIV Env-Gag fusion peptide and the other derived from a non-HIV-1 viral antigen (rubeola virus), did not produce any observable effect on the NK cell activity of LGL, demonstrating the specificity of the effect produced by Env-Gag. Subsequent treatment of LGL with alpha interferon (IFN-alpha) or interleukin 2 (IL-2) alone partially reversed the Env-Gag-induced suppression of NK cell activity. However, LGL treated with both IFN-alpha and IL-2 completely reversed the suppression of NK cell cytotoxicity by Env-Gag. The combined effect of IFN-alpha and IL-2 in enhancing NK cell activity may provide a novel therapeutic approach to the restoration of depressed NK cell activity observed in HIV-infected patients.     

Pretreatment of human vascular smooth muscle cells with interleukin-1 enhances interleukin-6 production and cell proliferation (action of IL-1 on vascular smooth muscle cells)

Systemic vasculitis is an inflammatory disorder of blood vessels characterized by a perivascular mononuclear cell infiltration around the vessel and fibrinoid necrosis within vessel walls. Interleukin-1 (IL-1) is a multipotent inflammatory mediator and affects several properties of vascular cells. To determine whether IL-1 could contribute to the pathogenesis of vascular diseases, we examined the effect of IL-1 on B cell stimulatory factor-2/interleukin-6 (IL-6) production by cultured human vascular smooth muscle cells (SMC) and the proliferation of these cells. Supernatants of SMC stimulated IgM synthesis of human B cell line. SKW6-CL4 cells. This activity was increased (1.7 to 2.6-fold) when SMC were pretreated with IL-1 or calcium ionophore A23187 for 48 h, and was completely blocked by rabbit anti-human IL-6 antibodies. These IL-6 activities of the SMC supernatants were also assessed by using an IL-6 dependent murine hybridoma cell line. MH-60. BSF-2. In addition, we observed that pretreatment of SMC with IL-1 for 48 h stimulated growth of SMC during the 96 h incubations, as assessed by cell number (p less than 0.05). These results suggest that IL-1 may contribute to the pathogenesis of inflammatory and immunological vasculitis by the augmentation of IL-6 release and growth of SMC.