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The efficiency of immune-mediated clearance of cancer cells is hampered by immunosuppressive mediators in the malignant microenvironment, including NADPH oxidase-derived reactive oxygen species. We aimed at defining the effects of histamine, an inhibitor of the myeloid NADPH oxidase/NOX2, on the development of Ag-presenting dendritic cells (DCs) from myeloid precursors and the impact of these mechanisms for tumor growth. Histamine was found to promote the maturation of human DCs from monocytes by increasing the expression of HLA-DR and costimulatory molecules, which resulted in improved induction of Th cells with Th0 polarity. Experiments using wild-type and NOX2-deficient myelomonoblastic cells showed that histamine facilitated myeloid cell maturation only in cells capable of generating reactive oxygen species. Treatment of mice with histamine reduced the growth of murine EL-4 lymphomas in parallel with an increment of tumor-infiltrating DCs in NOX2-sufficient mice but not in NOX2-deficient (gp91(phox) (-/-)) mice. We propose that strategies to target the myeloid NADPH oxidase may facilitate the development of endogenous DCs in cancer., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Phagocyte-derived reactive oxygen species ("oxygen radicals") have been ascribed a suppressive role in immunoregulation by inducing dysfunction and apoptotic cell death in lymphocytes. Earlier studies show that human NK cells are exceptionally sensitive to oxygen radical-induced apoptosis and functional inhibition. Two subsets of human CD56(+) NK cells have been identified: the highly cytotoxic CD56(dim) cells which constitute >90% of NK cells in peripheral blood, and the less cytotoxic but efficiently cytokine-producing CD56(bright) cells. In this study, we demonstrate that the CD56(bright) subset of NK cells, in contrast to CD56(dim) cells, remains viable and functionally intact after exposure to phagocyte-derived or exogenously added oxygen radicals. The resistance of CD56(bright) cells to oxidative stress was accompanied by a high capacity of neutralizing exogenous hydrogen peroxide, and by a high cell-surface expression of antioxidative thiols. Our results imply that CD56(bright) NK cells are endowed with an efficient antioxidative defense system that protects them from oxygen radical-induced inactivation.

Human NK cells (with CD3-/56+ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology, degradation of DNA into oligonucleosomal fragments, and reduced nuclear interchalation of propidium iodide. In contrast, T cells (CD3+/56-) remained non-apoptotic. The monocyte-induced apoptosis in NK cells was prevented by catalase, a scavenger of hydrogen peroxide; whereas superoxide dismutase (a scavenger of superoxide anion), hydroxyl radical scavengers such as mannitol and deferoxamine, or the hypochlorus acid scavenger taurine did not prevent apoptosis. Sodium azide, a myeloperoxidase inhibitor, substantially reduced the monocyte-induced apoptosis in NK cells. Exogenous hydrogen peroxide, at concentrations exceeding 1 microns, induced apoptosis in both NK and T cells. Apoptosis induced by hydrogen peroxide occurred independently of synthesis of protein or mRNA and was blocked by the endonuclease inhibitor aurin tricarboxylic acid. Furthermore, oxidatively induced apoptosis in NK cells was inhibited by herbimycin A, indicating that apoptosis was dependent on protein kinases. Two to five times more hydrogen peroxide was required to induce apoptosis in T cells compared with NK cells. Similarly, NK cells were considerably more susceptible to apoptosis induced by the topoisomerase II inhibitor etoposide or by gamma-irradiation than were T cells. We conclude that monocyte-derived reactive oxygen metabolites kill NK cells by apoptosis and that NK cells are unusually sensitive to oxidatively as well as non-oxidatively induced apoptosis.

Monocytes, recovered by centrifugal elutriation, effectively inhibit functions of human NK cells in vitro. Histamine, acting via monocyte H2-type histamine receptors, abrogates the inhibitory signal. The aim of this study was to define the histamine-reversible mechanism by which monocytes inhibit NK cells with special reference to the respiratory burst activity of monocytes. Monocytes recovered from patients with chronic granulomatous disease did not suppress NK cell function, indicating the requirement of intact nicotinamide-adenine dinucleotide phosphate (NAPDH) oxidase activity of monocytes to inhibit NK cells. Furthermore, catalase, a scavenger of hydrogen peroxide (H2O2), was found to potently reverse the monocyte-induced inhibition of NK cell function; on the other hand, superoxide dismutase (a scavenger of superoxide anion), hydroxyl radical scavengers such as mannitol and deferoxamine, the hypochlorous acid scavenger taurin, or the nitric oxide synthetase inhibitor NG-monomethyl-L-arginine (L-NMMA) did not affect the monocyte-derived, suppressive signal. H2O2, at micromolar concentrations, reconstituted the inhibitory effects of monocytes on NK cell function. Histamine had no scavenger activity but effectively suppressed the generation of H2O2 in isolated monocytes. This effect of histamine was transduced by H2-type histamine receptors. We conclude that the histamine-reversible, inhibitory effect of elutriated monocytes on NK cells is dependent on the formation of reactive oxygen metabolites by monocytes and that H2O2 is a pivotal mediator of the suppressive signal.

The formation of lung metastases by i.v.-injected B16 melanoma (F1 and F10 strain) cells in Swiss albino, C57BL/6, and BALB/c mice was reduced by a single dose of histamine given 24 h before tumor cell inoculation. The antimetastatic effect of histamine was specifically mediated by histamine H2-receptors (H2R): it was blocked by the H2R antagonist ranitidine and mimicked by dimaprit, a specific H2R agonist but not by an H2R-inactive structural analog of this compound, nor-dimaprit, or the H1R agonist 2-thiazolyl-ethylamide. A single dose of any of the H2R antagonists ranitidine, tiotidine, famotidine, or cimetidine drastically augmented metastasis. Effects of H2R-interactive compounds on B16 metastasis required intact NK cells, as judged by the inability of histamine or ranitidine to affect B16 metastasis after NK cell depletion in vivo using antibodies to asialo-GM1. NK-cell-mediated lysis of YAC-1 lymphoma cells in vivo was enhanced by histamine and reduced by ranitidine within 4 h after inoculation of tumor cells. The antimetastatic effect of IL-2 was potentiated by histamine; in some experiments, combined treatment with a low dose of IL-2 (6000 U/kg) and histamine completely eliminated metastasis, whereas concomitant treatment with ranitidine abrogated antimetastatic effects of IL-2; animals treated with ranitidine and IL-2 displayed the same level of enhanced metastasis as those treated with ranitidine alone. The presented data are suggestive of an earlier unrecognized role for histamine in NK cell-mediated resistance against metastatic tumor cells.

Pretreatment of lymphocytes (16 hr, 37 degrees C) with adrenaline at final concentrations of 10(-7) to 10(-9) M, followed by removal of the drug, increased natural killer (NK) cell activity vs K562 leukemic cells in a 4-hr 51Cr-release assay. The most efficient concentration of adrenaline was 10(-8) M; mean increase of NK activity over base-line activity for all donors examined was 30%. However, the individual response to adrenaline pretreatment was variable; in some donors, the effect was equal to maximal interferon (IFN) stimulation. Effects of adrenaline pretreatment were consistently reduced to base-line activity by co-incubation with the nonselective beta-adrenoceptor antagonist propranolol at 100-fold higher concentrations. The enhancing effect of adrenaline (10(-8) M) pretreatment was also observed after 1-hr pretreatment; this effect was prevented by simultaneous incubation with propranolol but was not affected by dex-propranolol. Direct addition of adrenaline to lymphocyte/target cell mixtures was inhibitory at 10(-6) M adrenaline concentration. The inhibitory effect of adrenaline in this assay was again completely prevented by propranolol and unaffected by dex-propranolol. The observed stimulatory effect of adrenaline pretreatment could not be ascribed to IFN production. Data presented indicate a dual effect of adrenaline on NK cell activity and suggest both a positive and a negative beta-adrenoceptor-mediated regulation of human NK cells.

We have investigated effects of histamine on the spontaneous cytotoxic activity of human natural killer (NK) cells in vitro. Addition of histamine (10(-3) to 10(-7) M) to assay cultures of Percoll-fractionated mononuclear cells (MNC) and erythroleukemic K 562 target cells resulted in a strong enhancement of the cytotoxicity of low-density MNC, enriched for NK cell cytotoxicity (NKCC). No enhancing or suppressing effects of histamine could be detected after removal of monocytes/adherent cells from the effector cell suspensions. When unfractionated MNC were used as NK effectors, similar results were obtained, i.e., dose-dependent enhancement of NKCC by histamine in the presence of monocytes and lack of effect in nonadherent effector cells. Freshly isolated monocytes displayed low spontaneous cytotoxicity against K 562 targets and were not induced by histamine. The histamine-induced enhancement was mimicked by dimaprit, a specific histamine H2-receptor agonist, but not by N-methyldimaprit, a chemical control for H2-receptor agonist activity of dimaprit. Furthermore, the enhancement was completely antagonized by the specific histamine H2-receptor antagonists cimetidine and ranitidine. The effect of histamine could not be ascribed to endogenous interferon (IFN) production, since no IFN activity could be detected in histamine-treated MNC effectors. Also, the enhancing effects of histamine and human leukocyte IFN-alpha were clearly additive. On the basis of these findings, we suggest that histamine, via specific activation of H2 receptors, may be an important regulator of human NK cell activity.

Addition of serotonin to mixtures of target cells and natural killer (NK)-enriched human mononuclear cells (MNC) in a 4-hr 51Cr-release assay strongly augmented NK cell cytotoxicity (NKCC) vs K562, Chang, or Molt-4 target cells. The effect was dose dependent at serotonin concentrations of 10(-4) to 10(-7) M, expressed at several effector to target cell ratios, and required the presence of accessory monocytes. A 5-HT1-specific receptor agonist, 8-OH-DPAT, mimicked the enhancing properties of serotonin with similar potency. Equimolar concentrations of the mixed 5-HT1/5-HT2 receptor antagonist cyproheptadine, but not the 5-HT2-specific antagonist ketanserin, completely blocked the serotonin-induced NKCC enhancement. Monocyte/NK cell mixtures incubated with serotonin for 1 hr produced a soluble factor that could enhance the cytotoxicity of autologous, NK-enriched cells depleted of monocytes, which did not respond to serotonin alone. The factor displayed no IFN or IL 2 activity as judged by the lack of antiviral activity and inability to support the growth of an IL 2-dependent cell line. In the presence of monocytes, serotonin (10(-5) M) was considerably more effective than human IFN-alpha or IFN-gamma at optimal concentrations and was about equally effective as IL 2 at a final concentration of 50 U/ml in a short-term NK assay. The potency and efficacy for serotonin were similar to that earlier reported for histamine in monocyte-containing effector cells. The NKCC-enhancing effect of serotonin was additive to that induced by IFN-alpha, IFN-gamma, or IL 2, but not to histamine. The presented data suggest an earlier unrecognized, serotonin receptor-mediated regulation of human NK cells.