Human monocytes are stimulated for nitric oxide release in vitro by some tumor cells but not by cytokines and lipopolysaccharide

Nitric oxide (NO) has been recently identified as a potent mediator of tumoricidal activity of activated macrophages. Macrophages can be activated for tumor cell killing by microbial products, including lipopolysaccharide (LPS) and various cytokines. Here we report that in contrast to mouse macrophages, human peripheral blood monocytes stimulated with cytokines or LPS failed to release NO. Also priming of monocytes with interferon-γ followed by activation with cytokines or LPS did not cause NO secretion. However, monocytes responded with NO production to stimulation with some human cancer cells but not with untransformed cells. NO production by monocytes was inhibited by N^G-monomethyl-L-arginine, specific inhibitor of NO synthase and emetine, an irreversible blocker of protein synthesis. This may imply that human monocytes are unique in their restricted capacity to produce NO following interaction with some tumor cells, but not with other stimulators, and in this respect they may be able to distinguish between malignant and normal cells.     

Characteristics of bacterial lipopolysaccharide induction of interleukin 1 synthesis and secretion by human monocytes.

The objective of these studies was to characterize some aspects of interleukin 1 (IL-1) synthesis and secretion by human monocytes after stimulation with bacterial lipopolysaccharides (LPS). Various molecular species of LPS were incubated with adherent monocytes for 24 h. IL-1 activity in monocyte supernatants (secretion) and lysates (synthesis) was determined by stimulation of collagenase production in rabbit articular chondrocytes and augmentation of mitogen-induced proliferation of murine thymocytes. The presence of cytochalasin B enhanced LPS-induced IL-1 secretion without altering IL-1 synthesis. Monocytes preincubated in dexamethasone or hydrocortisone failed to exhibit any IL-1 activity in supernatants after LPS stimulation but the cell lysates still possessed 50% of control IL-1 activity. Studies with different LPS preparations indicated that the presence of diphosphoryl groups in lipid A enhanced the IL-1-inducing activities. Butanol-extracted LPS preparations, containing associated proteins, were not completely inhibited by 5 micrograms/ml polymyxin B in induction of IL-1 production at LPS concentrations of 10 or 100 ng/ml. These results indicate that the failure of polymyxin B to inhibit stimulation of IL-1 production by tests materials cannot be assumed to mean an absence of contaminating LPS.     

Synergistic activation of human T cells by interleukin 1 and interleukin 6

Purified human interleukin 6 (IL 6) was found to stimulate the proliferation of human tonsillar and peripheral rosetting T cells subliminally activated with phytohemagglutinin (PHA). This response seemed independent of IL 2 but highly dependent on the presence of accessory cells. Indeed, when accessory cell-depleted tonsillar T cells were activated with PHA and exposed to IL 6, only minimal proliferations were observed. A similar result was obtained with IL 1. However, a combination of these two cytokines induced strong proliferations, indicating that IL 1 and IL 6 plays a synergistic role in the interactions between accessory cells and T lymphocytes.     

Trogocytosis and killing of IL-4-polarized monocytes by autologous NK cells

Cross-regulations between innate immune cells have been given more and more emphasis. Here, we address the question of bidirectional interactions between activated monocytes and autologous NK cells. Classically activated monocytes (class-monocytes), obtained by priming with IFN-gamma, drive an inflammatory immune response. On the contrary, alternatively activated monocytes (alt-monocytes), obtained by stimulation with IL-4 or IL-13, engage an anti-inflammatory immune response. We show that alt-monocytes inhibit proliferation and production of IFN-gamma by autologous, IL-2-activated NK cells, whereas class-monocytes do not inhibit these NK cell functions. Reciprocally, IL-2-activated NK cells interact and undertake intensive synaptic transfer with alt-monocytes, whereas interactions with class-monocytes are weaker. This strong trogocytosis correlates with an efficient killing of alt-monocytes, mediated by natural cytotoxicity receptors and a lowered killing of class-monocytes. These results suggest that interactions between NK cells and autologous-activated monocytes modulate inflammatory responses. This might be extended further in the elimination of tumor-associated macrophages, which actively promote solid tumor progression and metastasis.     

Human mast cells present antigen to autologous CD4+ T cells

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CXCR4 expression on monocytes is up-regulated by dexamethasone and is modulated by autologous CD3+ T cells

Chemokines and their receptors regulate cell migration to sites of inflammation. The glucocorticoid dexamethasone has potent anti-inflammatory effects, yet paradoxically up-regulates expression of some cytokine receptors. We have examined the effects of dexamethasone on chemokine receptor expression. Using an RNase protection assay, we show that dexamethasone up-regulates human peripheral blood mononuclear cell (PBMC) expression of CXCR4 mRNA. Flow cytometric analysis demonstrated that increased expression of CXCR4, but not CXCR1 and CXCR2, occurred on both monocytes and CD3+ T cells in PBMC mixed cultures. A stromal-derived factor (SDF)-1alpha-mediated calcium influx was detected on monocytes. Basal levels of CXCR4 expression on purified monocytes were lower when compared with monocytes in mixed PBMC cultures. Co-culture of monocytes with purified CD3+ T cells led to enhanced basal expression of CXCR4 on monocytes. The use of transwells to partition CD3+ T cells resulted in increased CXCR4 expression on monocytes, suggesting that CD3+ T-cell derived soluble factors regulate CXCR4 expression.     

C5a-mediated release of interleukin 6 by human monocytes

Recombinant human C5a (rC5a) was assessed for its ability to induce interleukin 6 (IL-6) production in human peripheral blood-derived mononuclear cell (PBMC) cultures. rC5a was observed to induce IL-6 production as measured by hybridoma growth promotion (B9.9 assay) and human B cell differentiation (SKW6.4 assay). Optimal IL-6 production was obtained after 24 hr stimulation with 0.1-0.5 micrograms/ml rC5a. In addition, natural human C5ades Arg and natural porcine C5a were able to induce a similar level of IL-6. The observed IL-6 activity appeared not to be due to endotoxin contamination since heat treatment (100 degrees C/15 min) inhibited rC5a induction of IL-6. The rC5a stimulation also induced an increase in steady-state IL-6 mRNA as determined by Northern blot analysis. Pretreatment of PBMC with leucine-methyl ester to deplete monocytes reduced the rC5a-induced IL-6 production to background levels. In addition, stimulation of purified T cell preparations with rC5a produced little IL-6 activity, suggesting that monocytes are the major source of IL-6 in this system. These results suggest that the inflammatory and immunoregulatory activities of C5a may in part be due to the stimulation of IL-6 release, a cytokine which possesses potent pleiotropic functions.     

Constitutive secretion of interleukin 1 by human monocytes

The constitutive and lipopolysaccharide (LPS)-induced secretion of interleukin 1 (IL1) by cultured human monocytes and macrophages has been studied. Both freshly obtained monocytes and their culture-derived macrophages were induced by LPS to secrete similar amounts of IL1. Such induction, however, was accompanied by the secretion of dialyzed inhibitory activity. Constitutive secretion of IL1 was detected in concentrated supernatants of monocyte cultures. The factor obtained constitutively did not manifest significant inhibitory activity. A method is described for the recovery of IL1-containing supernatants in serum- and other stimulant-free medium. The biological activities of the constitutively secreted IL1 were similar to the LPS-induced activities. The constitutive secretion of IL1 was not equally distributed in the entire monocyte population. We found that a small fraction of loosely adherent monocytes secreted higher amounts of IL1 than the strongly adherent monocytes. However, the property of higher secretion of IL1 was not stable and disappeared following monocyte cultivation. Thus, constitutive activity of IL1 could be recovered either by concentrating the culture supernatants or by enriching a subset of monocytes with higher IL1 activity.     

白介素18在脂多糖致大鼠脑水肿中的表达

目的探讨白介素18（IL-18）在脂多糖（LPS）致大鼠脑水肿发病过程中的表达及纳络酮对其干预作用。方法SD大鼠84只，对照组（NS组）：28只，0．2mL生理盐水颈内动脉注射；内毒素组（LPS组）：28只，颈内动脉注射LPS 200μg；纳络酮治疗组（NAL组），28只，颈内动脉注射LPS后10min、1、2、6、12h及处死前2h腹腔注射纳络酮1mg／kg。于不同时间点测定脑组织匀浆IL-18的含量。干湿法测定脑组织含水量，甲酰胺法测定伊文思兰（EB）含量。结果LPS组脑组织含水量和EB含量显著高于NS组（P〈0．01）。NAL组脑组织含水量和EB含量显著低于LPS组（P〈0．01），但仍较NS组高（P〈0．01）。LPS组IL-18的含量显著高于NS组（P〈0．01）。NAL组IL-18在4、6、12h时表达降低，与LPS组比较差异显著（P〈0．05或P〈0．01），但在48h时差异无显著性（P〉0．05）。LPS组脑组织含水量和EB含量呈正相关（r=0．743，P〈0．01），IL-18含量与含水量呈正相关（r=0．616，P〈0．01），IL-18含量与EB含量呈正相关（r=0．497，P〈0．01）。结论IL-18参与了脑水肿的发生发展，纳络酮可以抑制IL-18的生成，减轻脑水肿。     

Characterization of biological response modifier release by human monocytes cultured in suspension in serum-free medium

Human monocytes have been shown to be critical immunoregulatory cells for a variety of frequently measured in vitro human immune functions and are secretors of potent biologic response modifiers (BRMs). These BRMs, such as interferon (IFN), colony stimulating factor (CSF) and prostaglandin E (PGE), may play important roles in the host immune response to cancer. A new approach for culturing circulating peripheral blood monocytes has been developed to retain the native characteristics of these cells, avoiding possible alteration or activation by adherence. The ability of elutriator-purified human monocytes to secrete IFN and PGE was examined under conditions of suspension culture as well as after adherence, and no difference in secretion of these BRMs was noted. In contrast, Teflon-cultured monocytes demonstrated a significantly enhanced CSF release over culturing in polystyrene plates. A new serum-free medium has also been developed, and monocytes cultured in vitro in this medium showed a 5-fold increase in IFN release, and up to a 72% increase in CSF release when compared to optimal standard culture medium (containing 10% AB serum) and an increased stimulation index for PGE release. By these procedures, hundreds of millions of highly purified human monocytes can be sterilely isolated in suspension and cultured in suspension in serum-free medium with retention of BRM-releasing capabilities. This system should permit more detailed molecular studies of monocytes (in which serum can be an impediment) and may also facilitate clinical therapy studies involving the in vivo transfer of monocytes activated in suspension in vitro.     

Human T lymphocyte priming in vitro by haptenated autologous dendritic cells.

Dendritic cells (DC), generated from adherent peripheral blood mononuclear cells (PBMC) by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, were used to study in vitro sensitization of naive, hapten-specific T cells and to analyse cross-reactivities to related compounds. DC were hapten-derivatized with nickel sulphate (Ni) or 2-hydroxyethyl-methacrylate (HEMA), followed by tumour necrosis factor-alpha (TNF-alpha)-induced maturation, before autologous T cells and a cytokine cocktail of IL-1beta, IL-2 and IL-7 were added. After T cell priming for 7 days, wells were split and challenged for another 7 days with Ni or HEMA, and potentially cross-reactive haptens. Hapten-specificity of in vitro priming was demonstrated by proliferative responses to the haptens used for priming but not to the unrelated haptens. Highest priming efficiencies were obtained when both IL-4 and IL-12 were added to the cytokine supplement. Marked interferon-gamma (IFN-gamma) release (up to 4 ng/ml) was found when IL-12 was included in the cultures, whereas IL-5 release (up to 500 pg/ml) was observed after addition of IL-4 alone, or in combination with IL-12. Nickel-primed T cells showed frequent cross-reactivities with other metals closely positioned in the periodic table, i.e. palladium and copper, whereas HEMA-primed T cells showed distinct cross-reactivities with selected methacrylate congeners. Similar cross-reactivities are known to occur in allergic patients. Thus, in vitro T cell priming provides a promising tool for studying factors regulating cytokine synthesis, and cross-reactivity patterns of hapten-specific T cells.     

Cyclosporin A mediates immunosuppression of primary cytotoxic T cell responses by impairing the release of interleukin 1 and interleukin 2

The site of action of the immunosuppressive drug cyclosporin A in in vitro cytotoxic allograft responses has been localized. General cytotoxic effects of the drug on proliferating T cells became apparent at concentrations of 500-1000 ng/ml, while selective effects were observed at concentrations of 10-100 ng/ml. The selective effects included a blockade of interleukin 2 release from activated T helper cells on the one hand and inhibition of interleukin 1 release from splenic adherent cells on the other. While cyclosporin A did not interfere with the intracellular events required for the activation and subsequent clonal expansion of alloreactive T cells, the lack of interleukin 1 and interleukin 2 induced by cyclosporin A results in an inability of T responder cells to mount cytotoxic allograft responses in vitro.     

Antigen-specific CD8+ T cells inhibit IgE responses and interleukin-4 production by CD4+ T cells

There is a growing body of evidence which suggests that CD8⁺ T cells play an important part in regulating the IgE response to non-replicating antigens. In this study we have systematically investigated their role in the regulation of IgE and of CD4⁺ T cell responses to ovalbumin (OVA) by CD8⁺ T cell depletion in vivo. Following intraperitoneal immunization with alum-precipitated OVA, OVA-specific T cell responses were detected in the spleen and depletion of CD8⁺ T cells in vitro significantly enhanced the proliferative response to OVA. Depletion of CD8⁺ T cells in vivo 7 days after immunization failed to enhance IgE production, while depletion of CD8⁺ T cells on days 12–18 greatly enhanced the IgE response, which rose to 26 μ/ml following a second injection of anti-CD8 on day 35 and remained in excess of 1 μ/ml over 300 days afterwards. Reconstitution on day 21 of rats CD8-depleted on day 12 with purified CD8⁺ T cells from animals immunized on day 12 completely inhib ited the IgE response. This effect was antigen specific; CD8⁺ T cells from OVA-primed animals had little effect on the IgE response of bovine serum albumin immunized rats. In vivo, CD8⁺ T cell depletion decreased interferon (IFN)-γ production but enhanced interleukin (IL)-4 production by OVA-stimulated splenic CD4⁺ T cells. Furthermore, CD8⁺ T cell depletion and addition of anti-IFN-γ antibody enhanced IgE production in vitro in an IL-4-supplemented mixed lymphocyte reaction. These data clearly show that antigen-specific CD8⁺ T cells inhibit IgE in the immune response to non-replicating antigens. The data indicate two possible mechanisms: first, CD8⁺ T cells have direct inhibitory effects on switching to IgE in B cells and second, they inhibit OVA-specific IL-4 production but enhance IFN-γ production by CD4⁺ T cells.     

Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells,but not on T cells

Interleukin 13 (IL-13) is a recently described protein secreted by activated T cells which is a potent in vitro modulator of human monocyte and B-cell functions. The data, reviewed here by Gerard Zurawski and Jan de Vries, shows that IL-13 shares biological activities with IL-4, their genes are closely linked in both the human and mouse genomes, and there is sequence homology between IL-13 and IL-4 proteins. Although the cloned IL-4 receptor protein (IL-4R) does not bind IL-13, it appears that the functional IL-4R and IL-13R share a common subunit that is important for signal transduction.     

Human lymphocytes induce a differential release of prostaglandin E2 and interleukin 1-like mononuclear cell factor from normal blood monocytes

Lymphocytes influence the production of both prostaglandin E2 (PGE2) and interleukin 1 (IL 1) by monocytes. We have examined whether these two products are released concomitantly or not under identical culture conditions using monocyte- and lymphocyte-enriched populations obtained on Percoll gradient. IL 1 was measured as mononuclear cell factor (IL 1/MCF). When incubated with concanavalin A, the monocyte-enriched fraction (MF; 80-91% monocytes), but not the lymphocyte-enriched fraction (LF; 95% lymphocytes) produced increasing amounts of PGE2 and MCF. However, when LF cells were added to MF cells in culture, a 40% to 60% decrease of PGE2 secretion was observed whereas the MCF production remained unchanged or increased up to 26-fold. Such a dissociation between IL 1 and PGE2 production by monocytes indicates independent regulation mechanisms in controlling the immune response under the influence of lymphocytes.     

Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages.

Lipopolysaccharide (LPS) is a potent inducer of interleukin 1 (IL 1) synthesis and release, and of tumor necrosis factor (TNF) secretion. Many signals can enhance the LPS-induced production of these cytokines. We have previously observed that addition of low amounts of normal human serum to the culture medium enhances IL 1 production. Among serum factors, anaphylatoxins C3a and C5a and/or their desArg derivatives have been shown to enhance LPS-induced IL 1 and TNF production. However, the capacity of natural anaphylatoxins to induce by themselves the production of cytokines remains a controversial issue. We have investigated the capacity of human recombinant C5a (hrC5a) to induce IL 1 and TNF production. Despite its lack of direct triggering, hrC5a was able to act synergistically with LPS, leading to higher IL 1 and TNF release by human monocytes and mouse peritoneal macrophages. As assessed by the comitogenic assay, hrC5a increased IL 1 release, whereas cell-associated IL 1 activity was not significantly modified. Measurement by enzyme-linked immunosorbent assay of human IL 1 beta led to similar conclusions, whereas measurement of IL 1 alpha by radioimmunoassay indicated, in addition, an increase in intracellular IL 1 alpha.     

Adenosine triphosphate affects interleukin -1beta release by T98G glioblastoma cells through a purinoceptor-independent mechanism

T98G glioblastoma cells were previously shown to significantly increase interleukin-1beta (IL-1beta) mRNA levels in response to IL-1beta stimulation. This work demonstrates that in such conditions T98G, despite possessing biologically active interleukin converting enzyme, do not release detectable amounts of IL-1beta, even in the presence of 20 mM adenosine triphosphate (ATP). IL-1beta secretion is observed only following concomitant stimulation with 1000 units/ml of IL-1beta and 20 mM ATP. ATP induces a dose-dependent depolarization of T98G plasma membrane, whereas it does not affect Ca(2+) concentration or cell membrane permeability. Our data, together with the observation that the depolarizing effects of ATP are retained after preincubation with 100 microM suramin, an antagonist of P2-purinoceptors, suggest that ATP plays a role in IL-1beta secretion by T98G but its effects do not occur through P2-purinoceptors.