Immune enhancement and anti-tumour activity of IL-23

Immunotherapy, including the use of cytokines and/or modified tumour cells immune stimulatory cytokines, can enhance the host anti-tumour immune responses. Interleukin-23 (IL-23) is a relative novel cytokine, which consists of a heterodimer of the IL-12p40 subunit and a novel p19 subunit. IL-23 has biological activities similar to but distinct from IL-12. IL-23 can enhance the proliferation of memory T cells and the production of IFN-γ, IL-12 and TNF-α from activated T cells. IL-23 activates macrophages to produce TNF-α and nitric oxide. IL-23 can also act directly on dendritic cells and possesses potent anti-tumour and anti-metastatic activity in murine models of cancer. IL-23 can also induce a lower level of IFN-γ production compared with that induced by IL-12. This may make IL-23 an alternative and safer therapeutic agent for cancer, as IL-12 administration can lead to severe toxic side effects because of the extremely high levels of IFN-γ it induces.This article is a symposium paper from the Annual Meeting of the “International Society for Cell and Gene Therapy of Cancer”, held in Shenzhen, China, on 9–11 December 2005.     

Augmentation of antigen-presenting and Th1-promoting functions of dendritic cells by WSX-1(IL-27R) deficiency

WSX-1 is the alpha subunit of the IL-27R complex expressed by T, B, NK/NKT cells, as well as macrophages and dendritic cells (DCs). Although it has been shown that IL-27 has both stimulatory and inhibitory effects on T cells, little is known on the role of IL-27/WSX-1 on DCs. LPS stimulation of splenic DCs in vivo resulted in prolonged CD80/CD86 expression on WSX-1-deficient DCs over wild-type DCs. Upon LPS stimulation in vitro, WSX-1-deficient DCs expressed Th1-promoting molecules higher than wild-type DCs. In an allogeneic MLR assay, WSX-1-deficient DCs were more potent than wild-type DCs in the induction of proliferation of and IFN-gamma production by responder cell proliferation. When cocultured with purified NK cells, WSX-1-deficient DCs induced higher IFN-gamma production and killing activity of NK cells than wild-type DCs. As such, Ag-pulsed WSX-1-deficient DCs induced Th1-biased strong immune responses over wild-type DCs when transferred in vivo. WSX-1-deficient DCs were hyperreactive to LPS stimulation as compared with wild-type DCs by cytokine production. IL-27 suppressed LPS-induced CD80/86 expression and cytokine production by DCs in vitro. Thus, our study demonstrated that IL-27/WSX-1 signaling potently down-regulates APC function and Th1-promoting function of DCs to modulate overall immune responses.     

Induction of interleukin-1 and tumor necrosis factor alpha in brain cultures by human immunodeficiency virus type 1.

Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) are produced by leukocytes and play a role in immune responses. They also function in normal brain physiology as well as in pathological conditions within the central nervous system, where they are produced by brain macrophages (microglia) and brain astrocytes. In this study, we document the ability of human immunodeficiency virus type 1 (HIV-1) to induce TNF alpha and IL-1 in primary rat brain cultures. While productive infection did not occur in these cells, it was not required for cytokine induction. Using monocyte/macrophage-tropic (JRFL) and T-cell-tropic (IIIB) strains of HIV-1, we were able to induce cytokines in both microglia and astrocytes. In addition to whole virus, recombinant envelope proteins also induced these cytokines. The induction of IL-1 and TNF alpha could be blocked by a panel of antibodies recognizing epitopes in the gp120 and gp41 areas of the envelope. Soluble recombinant CD4 did not block TNF alpha and IL-1 production. If TNF alpha and IL-1 can be induced in brain tissue by HIV-1, they may contribute to some of the neurologic disorders associated with AIDS.     

Enhanced apoptotic cell clearance capacity and B cell survival factor production by IL-10-activated macrophages: implications for Burkitt's lymphoma

Burkitt's lymphoma (BL) is typified by frequent tumor cell apoptosis and significant macrophage infiltration. Since BL cells have an inherent tendency to undergo apoptosis at a high rate, we reasoned that macrophages in BL are functionally enhanced in at least two activities that have implications for tumor pathogenesis: 1) engulfment of apoptotic cells, an anti-inflammatory process known to suppress immune responses, and 2) production of BL cell survival factors that limit the extent of tumor cell apoptosis. In this study, we show that the microenvironment of BL is rich in the pleiotropic cytokine IL-10, which can be produced by both tumor cells and macrophages, and that IL-10-activated human macrophages have enhanced capacity to engulf apoptotic cells in vitro. This was found to be dependent on the macrophage tethering receptor of apoptotic cells, CD14. Furthermore, IL-10-activated macrophages were found to produce markedly higher levels of the B cell survival factor, B cell-activating factor of the TNF family/B lymphocyte stimulator (BAFF/BLyS) than macrophages matured in the absence of IL-10. Coculture of macrophages with BL cells further enhanced BAFF secretion. Significantly, we show that enhancement of BL cell survival by IL-10-activated macrophages is mediated by a BAFF-dependent component and that BAFF is produced at high levels by tumor-associated macrophages in situ. These results indicate that macrophages, regulated by IL-10, have the potential to promote BL pathogenesis, first, through suppression of antitumor immunity following enhanced engulfment of apoptotic tumor cells and, second, through increased production of tumor cell growth/survival factors.     

Estrogen receptor alpha (ERalpha) deficiency in macrophages results in increased stimulation of CD4+ T cells while 17beta-estradiol acts through ERalpha to increase IL-4 and GATA-3 expression in CD4+ T cells independent of antigen presentation

The effects of 17beta-estradiol (E2) on immune function have been extensively reported. The effects are dependent on concentration and duration of exposure and potential differences in signaling between the known E2 receptors, estrogen receptors (ER) alpha and ERbeta. Through the use of ER-deficient mice, we and others have begun to demonstrate the role of the two known receptors in modulating immune functional activities. Previous studies have shown that cells of the innate immune system have altered function (bactericidal capacity) and patterns of cytokine expression (increased proinflammatory cytokine expression) through amelioration of ERalpha signaling. In this study, we extend these studies to analysis of T cell differentiation and proliferation in APC-dependent and APC-independent in vitro assay systems. Our results demonstrate that ERalpha deficiency in splenic macrophages, but not CD11c+ splenic dendritic cells pulsed with OVA significantly enhances proliferative responses and IFN-gamma production by transgenic OVA peptide-specific (OT-II) CD4+ T cells when compared with Ag-pulsed APC from wild-type littermates. The addition of E2 in this culture system did not significantly affect the production of IFN-gamma. In addition, when purified CD4+ T cells from ERalpha-deficient and wild-type littermates were stimulated with anti-CD3/CD28 Ab in the absence of E2, there were no significant differences in IFN-gamma or IL-4 production. However, the addition of E2 significantly increased IL-4 secretion, as well as increased GATA-3 mRNA levels from ERalpha-replete CD4+ T cells, while this effect was abrogated in ERalpha-deficient CD4+ T cells.     

Activation, stimulation and uptake of bacterial ghosts in antigen presenting cells

Bacterial ghosts have been shown to be an innovative system to prepare vaccines of various bacteria with all features of the intact bacterial cell envelopes, especially all antigenic epitopes, but also to target recombinant proteins inserted in the cell envelopes of the ghost preparations to specific antigen presenting cells. To investigate the activation of the antigen presenting cell by bacterial ghosts in more detail we studied the uptake of bacterial ghosts in dendritic porcine cells and RAW macrophages and the induction of inflammatory mediators or mediators directing the immune response in THP-1 human macrophage cell line. The synthesis of inflammatory macrophage mediators such as TNFalpha in the THP1 cell line was stimulated by a hundred-fold higher dose of ghosts from Vibrio cholerae than the corresponding LPS using ELISA-analysis. These results confirm in vivo experiments indicating no toxic effects of ghosts in rabbits even after intravenous administration in doses stimulating significant humoral responses. We were also able to see a significant activation of IL-12 indicated by the analysis of IL-12(p70) synthesis and IL-12(p40) mRNA accumulation. This interleukine is of special importance in the activation of cellular TH1 immune responses. A rapid uptake of bacterial ghosts in macrophages within 10-30 min could be confirmed by electron microscopy. As antigen presentation is especially effective in porcine dendritic cells (DC) and even a low capacity of antigen uptake is sufficient for an induction of immune responses we investigated uptake and activation of bacterial ghosts by DC. DC are known to be phagocytic in specific immature stages. We found a significant uptake of bacterial ghosts from Actinobacillus pleuropneumoniae (App) and V. cholerae conjugated with FITC (fluorescinisothiocyanate) within 2 h. These data suggest that bacterial ghosts effectively stimulate monocytes and macrophages for the induction of TH1 directed immune responses and dendritic cells treated with bacterial ghosts may serve as a promising vehicle for active immunization and immunotherapy in situ.     

IL-10 inhibits lipopolysaccharide-induced CD40 gene expression through induction of suppressor of cytokine signaling-3

Costimulation between T cells and APCs is required for adaptive immune responses. CD40, an important costimulatory molecule, is expressed on a variety of cell types, including macrophages and microglia. The aberrant expression of CD40 is implicated in diseases including multiple sclerosis, rheumatoid arthritis, and Alzheimer's disease, and inhibition of CD40 signaling has beneficial effects in a number of animal models of autoimmune diseases. In this study, we discovered that IL-10, a cytokine with anti-inflammatory properties, inhibits LPS-induced CD40 gene expression. We previously demonstrated that LPS induction of CD40 in macrophages/microglia involves both NF-kappaB activation and LPS-induced production of IFN-beta, which subsequently activates STAT-1alpha. IL-10 inhibits LPS-induced IFN-beta gene expression and subsequent STAT-1alpha activation, but does not affect NF-kappaB activation. Our results also demonstrate that IL-10 inhibits LPS-induced recruitment of STAT-1alpha, RNA polymerase II, and the coactivators CREB binding protein and p300 to the CD40 promoter, as well as inhibiting permissive histone H3 acetylation (AcH3). IL-10 and LPS synergize to induce suppressor of cytokine signaling (SOCS)-3 gene expression in macrophages and microglia. Ectopic expression of SOCS-3 attenuates LPS-induced STAT activation, and inhibits LPS-induced CD40 gene expression, comparable to that seen by IL-10. These results indicate that SOCS-3 plays an important role in the negative regulation of LPS-induced CD40 gene expression by IL-10.     

IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.     

Type II-Activated Murine Macrophages Produce IL-4

Background

Type II activation of macrophages is known to support Th2 responses development; however, the role of Th2 cytokines (esp. IL-4) on type II activation is unknown. To assess whether the central Th2 cytokine IL-4 can alter type II activation of macrophages, we compared the ability of bone marrow-derived macrophages from wild type (WT) and IL-4Rα-deficient mice to be classically or type II-activated in vitro.

Results

We found that although both WT and IL-4Rα-deficient macrophages could be classically activated by LPS or type II activated by immune complexes plus LPS, IL-4Rα-deficient macrophages consistently produced much higher levels of IL-12p40 and IL-10 than WT macrophages. Additionally, we discovered that type II macrophages from both strains were capable of producing IL-4; however, this IL-4 was not responsible for the reduced IL-12p40 and IL-10 levels produced by WT mice. Instead, we found that derivation culture conditions (GM-CSF plus IL-3 versus M-CSF) could explain the different responses of BALB/c and IL-4Rα−/− macrophages, and these cytokines shaped the ensuing macrophage such that GM-CSF plus IL-3 promoted more IL-12 and IL-4 while M-CSF led to higher IL-10 production. Finally, we found that enhanced IL-4 production is characteristic of the type II activation state as other type II-activating products showed similar results.

Conclusions

Taken together, these results implicate type II activated macrophages as an important innate immune source of IL-4 that may play an important role in shaping adaptive immune responses.     

Endogenous interferon-alpha production by differentiating human monocytes regulates expression and function of the IL-2/IL-4 receptor gamma chain

In vitro monocyte-derived macrophages (MDMac) and synovial fluid macrophages from inflamed joints differ from monocytes in their responses to interleukin 4 (IL-4). While IL-4 can suppress LPS-induced interleukin beta (IL-beta) and tumour necrosis factor alpha (TNF-alpha) production by monocytes, IL-4 can suppress LPS-induced IL-1 beta, but not TNFalpha production by the more differentiated cells. Recently we reported a correlation between the ability of IL-4 to regulate TNFalpha production by monocytes and the expression of the IL-4 receptor gamma chain or gamma common (gamma c chain). Like MDMac, interferon alpha (IFNalpha)-treated monocytes expressed less IL-4 receptor gamma c chain, reduced levels of IL-4-activated STAT6 and IL-4 could not suppress LPS-induced TNFalpha production. In addition, like monocytes and MDMac, IFNalpha-treated monocytes expressed normal levels of the IL-4 receptor alpha chain and IL-4 significantly suppressed LPS-induced IL-1 beta production. With addition of IFNalpha-neutralizing antibodies, the ability of IL-4 to suppress LPS-induced TNFalpha production with prolonged monocyte culture was restored. Detection of IFNalpha in synovial fluids from inflamed joints further implicates IFNalpha in the inability of IL-4 to suppress TNFalpha production by synovial fluid macrophages. This study identifies a mechanism for the differential expression of gamma c and varied responses to IL-4 by human monocytes compared with MDMac.     

Involvement of nicotinic acetylcholine receptors in suppression of antimicrobial activity and cytokine responses of alveolar macrophages to Legionella pneumophila infection by nicotine.

Although nicotine is thought to be one of the major immunomodulatory components of cigarette smoking, how nicotine alters the host defense of the lung and, in particular, immune responses of alveolar macrophages, which are critical effector cells in the lung defense to infection, is poorly understood. Nicotinic acetylcholine receptors (nAChRs) are the receptor for nicotine and may be involved in the modulation of macrophage function by nicotine. In this study, therefore, nicotine-induced suppression of antimicrobial activity and cytokine responses of alveolar macrophages mediated by nAChRs to Legionella pneumophila, a causative agent for pneumonia, were examined. The murine MH-S alveolar macrophage cell line cells expressed the messages for alpha4 and beta2 subunits of nAChRs, but not alpha7 subunits, determined by RT-PCR. The nicotine treatment of MH-S alveolar macrophages after infection with L. pneumophila significantly enhanced the replication of bacteria in the macrophages and selectively down-regulated the production of IL-6, IL-12, and TNF-alpha, but not IL-10, induced by infection. These effects were completely blocked by a nonselective antagonist, d-tubocurarine, for nAChRs, but not by a selective antagonist, alpha-bungarotoxin, for alpha7-nAChRs. Furthermore, the stimulation of nAChRs with another agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, showed the same effects, which were blocked by the antagonist d-tubocurarine, on the bacterial replication and cytokine regulation with that of nicotine. Thus, the results revealed that nAChRs, the major exogenous ligands of which are nicotine, are involved in the regulation of macrophage immune function by nicotine and may contribute to the cigarette-induced risk factors for respiratory infections in smokers.     

Cytokine requirements for induction of systemic and mucosal CTL after nasal immunization.

Cholera toxin (CT) is frequently used as an experimental adjuvant intranasally for the induction of systemic and mucosal immunity. However, CT is highly reactogenic and not approved for use in humans. To define the cytokine requirements for the nasal activation of the systemic and mucosal immune system, and to design new adjuvants with efficacy similar to CT, we defined the cytokines that were able to replace CT as a nasal adjuvant for the induction of CTL. BALB/c mice were nasally immunized with an HIV immunogen that contains an MHC class I-restricted CTL epitope +/- cytokines and tested for HIV-specific immune responses. We found that combinations of IL-1alpha plus IL-18, IL-1alpha plus IL-12, and IL-1alpha plus IL-12 plus GM-CSF each induced optimal splenocyte anti-HIV CTL responses in immunized mice (range 60-71% peptide-specific (51)Cr release). Peak H-2D(d)-peptide tetramer-binding T cell responses induced by cytokine combinations were up to 5.5% of CD8(+) PBMC. Nasal immunization with HIV immunogen and IL-1alpha, IL-12, and GM-CSF also induced Ag-specific IFN-gamma-secreting cells in the draining cervical lymph node and the lung. The use of IL-1alpha, IL-12, and GM-CSF as nasal adjuvants was associated with an increased expression of MHC class II and B7.1 on nonlymphocytes within the nasal-associated lymphoid tissue/nasal mucosa. Thus, IL-1alpha, IL-12, IL-18, and GM-CSF are critical cytokines for the induction of systemic and mucosal CTL after nasal immunization. Moreover, these cytokines may serve as effective adjuvants for nasal vaccine delivery.