CD4 T cells producing pro-inflammatory interleukin-17 mediate high pathology in schistosomiasis

In murine schistosomiasis mansoni, pronounced CD4 T cell-mediated, egg-induced, hepato-intestinal immunopathology and death, whether genetically determined or elicited experimentally, are associated with failure to down-regulate a net pro-inflammatory immune response. Important evidence contributing to this notion comes from the observation that immunization with schistosome egg antigens in CFA (SEA/CFA) causes low pathology C57BL/6 mice to develop an exacerbated form of disease and death in a cytokine milieu characterized by elevated interferon (IFN)-gamma levels. Since such a pro-inflammatory environment presumes a signaling pathway involving interleukin (IL)-12, the SEA/CFA immunization model was used to examine the extent of hepatic immunopathology in the absence of this cytokine. Surprisingly, the IL-12p40 subunit was an absolute requirement for the development of exacerbated disease, whereas the IL-12p35 subunit was not. Moreover, significantly elevated in vitro production of IL-17, but not of IFN-gamma, correlated with the high pathology, and neutralization of IL-17 in vivo resulted in a significant reduction of hepatic inflammation. Our findings clearly demonstrate the pathogenic potential of the novel IL-17-producing T cell subpopulation (ThIL-17), previously shown to mediate chronic inflammation in autoimmune disease. They also imply that IL-23, but not IL-12, is the critical signal necessary to support the pro-inflammatory ThIL-17 subset involved in high pathology schistosomiasis.     

Praziquantel pharmacotherapy reduces systemic osteopontin levels and liver collagen content in murine schistosomiasis mansoni

The pathogenesis of schistosomiasis and the mechanism of disease regression after Praziquantel pharmacotherapy are not fully elucidated. Schistosoma mansoni egg antigens directly stimulate the expression of the profibrogenic molecule osteopontin (OPN), and systemic OPN levels strongly correlate with disease severity, suggesting its use as a potential morbidity biomarker. In this study, we investigated the impact of Praziquantel use on systemic OPN levels and on liver collagen deposition in chronic murine schistosomiasis. Praziquantel treatment significantly reduced systemic OPN levels and liver collagen deposition, indicating that OPN could be a reliable tool for monitoring PZQ efficacy and fibrosis regression in murine schistosomiasis.     

In vitro effects of cAMP-elevating agents and glucocorticoid either alone or in combination on the production of nitric oxide,interleukin-12 and interleukin-10 in IFN-gamma- and LPS-activated mouse peritoneal macrophages

The effects of cAMP-elevating agents,N ⁶-2′-O-dibutyryl cAMP (Bu₂cAMP), and glucocorticoid (dexamethasone) on the production of inflammatory mediators—nitric oxide and interleukin-12 (IL-12) and anti-inflammatory mediator interleukin-10 (IL-10) were demonstrated in murine peritoneal macrophages. Inducible nitric oxide synthase (iNOS) and iNOS mRNA were detected by northern blot and western blot, respectively. The cAMP elevating agents Bu₂cAMP and prostaglandin E₂ each alone did not show any effect on NO production but along with IFN-γ and lipolysaccharide (LPS) they slightly enhanced NO production. Dexamethasone inhibited NO production in IFN-γ-and LPS-treated cells; cAMP elevating agents interfered with the NO production inhibited by dexamethasone. Inhibition was revealed at the mRNA level as well as at protein level. Bu₂cAMP or dexamethasone either alone or synergistically inhibited IL-12 production; Bu₂cAMP interfered with dexamethasone-mediated inhibition of IL-10 production in IFN-γ-and LPS-treated macrophages. The use of glucocorticoids along with cAMP elevating agents was beneficial in lowering the level of inflammatory mediator IL-12 and producing high levels of the anti-inflammatory mediator IL-10 active in cell protection. On the other hand, inteference of Bu₂cAMP with dexamethasone-mediated NO inhibition may have adverse effect. Therefore, adverse effects due to cAMP-mediated interference (inhibition) with NO synthesis may occur in many inflammatory diseases during combined drug therapy by glucocorticoids and cAMP elevating agents.     

Inhibition of phosphodiesterase activity, airway inflammation and hyperresponsiveness by PDE4 inhibitor and glucocorticoid in a murine model of allergic asthma

Phosphodiesterase 4 (PDE4) isozyme plays important roles in inflammatory and immunomodulatory cells. In this study, piclamilast, a selective PDE4 inhibitor, was used to investigate the role of PDE4 in respiratory function and inflammation in a murine asthma model. Sensitized mice were challenged with aerosolized ovalbumin for 7 days, piclamilast (1, 3 and 10 mg/kg) and dexamethasone (2 mg/kg) were orally administered once daily during the period of challenge. Twenty-four hours after the last challenge, airway hyperresponsiveness to methacholine was determined by whole-body plethysmography, airway inflammation and mucus secretion by histomorphometry, pulmonary cAMP-PDE activity by HPLC, cytokine levels in bronchoalveolar lavage fluid and their mRNA expression in lung by ELISA and RT-PCR, respectively. In control mice, significant induction of cAMP-PDE activity was parallel to the increases of hyperresponsiveness, inflammatory cells, cytokine levels, mRNA expression as well as goblet cell hyperplasia. However, piclamilast dose-dependently and significantly improved airway resistance and dynamic compliance, and the maximal effect was similar to that of dexamethasone. Piclamilast treatment dose-dependently and significantly prevented the increase in inflammatory cell number and goblet cell hyperplasia, as well as production of cytokines, including eotaxin, TNFalpha and IL-4. Piclamilast exerted a weaker inhibitory effect than dexamethasone on eosinophils and neutrophils, had no effect on lymphocyte accumulation. Moreover, piclamilast inhibited up-regulation of cAMP-PDE activity and cytokine mRNA expression; the maximal inhibition of cAMP-PDE was greater than that exerted by dexamethasone, and was similar to dexamethasone on cytokine mRNA expression. This study suggests that inhibition of PDE4 by piclamilast robustly improves the pulmonary function, airway inflammation and goblet cell hyperplasia in murine allergenic asthma.     

The role of IL-12 in the control of MCMV is fundamentally different in mice with a retroviral immunodeficiency syndrome (MAIDS)

The present study investigates the susceptibility of C57BL mice exhibiting T cell immunodeficiency and lymphadenopathy induced by LP-BM5 murine leukaemia virus (MAIDS) to murine cytomegalovirus (MCMV). Treatment of normal (M-) mice with anti-IL-12 increased the contribution of IgG1 to the hypergammaglobulinaemia induced by MCMV, consistent with a shift towards a Th2 phenotype. This impaired control of early MCMV replication in the liver, with little effect in the spleen. Control of hepatic infection correlated with a vigorous splenic NK cytotoxic response in a subgroup of IL-12-depleted M- mice that remained healthy, while others became moribund. Mortality in IL-12-depleted MAIDS (M+) mice given MCMV was ultimately greater than in M- controls, but was delayed despite high levels of MCMV in the liver. IL-12 was required for optimal control of MCMV replication in M+ mice. This may involve cytotoxic activity because similar levels of infection were seen in bg/bg M+ mice, where the beige mutation impairs the formation of cytotoxic granules. Hence the ability of M+ mice to tolerate high titres of MCMV during acute infection may enable innate cytotoxic responses to clear MCMV. Interleukin-12 depletion of M- mice also increased salivary gland MCMV titres and depressed delayed-type hypersensitivity responses to MCMV antigen, normally mediated by CD4+ T cells. These changes were not observed in IL-12-depleted M+ mice.     

Acidic polysaccharides isolated from Phellinus linteus induce phenotypic and functional maturation of murine dendritic cells

Acidic polysaccharides (PL) isolated from Phellinus linteus are known to stimulate the proliferation of T lymphocytes and humoral immune functions to act as a polyclonal activator of B cells, and to inhibit tumor growth and metastasis. However, little is known about their immunomodulating effects or the effects of its mechanisms on murine bone marrow (BM)-derived dendritic cells (DC). In this study, it profoundly increased CD80, CD86, MHC I, and MHC II expression in murine, GM-CSF and IL-4 stimulated, BM-derived myeloid DC. The ability of unstimulated DC to uptake dextran was higher than that of PL- or LPS-stimulated DC. We analyzed the concentration of IL-12 secreted by DC using flow cytometry and ELISA. Untreated DC secreted a low concentration of IL-12, while PL- or LPS-stimulated DC secreted higher levels of IL-12 than untreated DC. There were no remarkable differences in the concentrations of IL-12 produced by PL- or LPS-stimulated DC. However, polymyxin B (PB; an LPS inhibitor) effectively inhibited the surface molecules and IL-12 production induced by LPS, but had no effect on the PL in DC. PL-treated DC were much more potent antigen-presenting cells in allogeneic immune response than untreated DC. PL treatment not only formed morphologically mature DC but also induced predominant migration to lymphoid tissues. Moreover, the inhibitors of protein tyrosine kinase (PTK) or protein kinase C (PKC) significantly blocked the expression of surface molecules and IL-12 production in PL-stimulated DC. Treatment of DC with PL directly induced PKC activity and phosphorylated PTK. Furthermore, CD11b and/or CD18 partially mediated PL-induced DC maturation.     

Dexamethasone up-regulates type II IL-1 receptor in mouse primary activated astrocytes

Brain astrocytes play a pivotal role in the brain response to inflammation. They express IL-1 receptors including the type I IL-1 receptor (IL-1RI) that transduces IL-1 signals in cooperation with the IL-1 receptor accessory protein (IL-1RAcP) and the type II IL-1 receptor (IL-1RII) that functions as a decoy receptor. As glucocorticoid receptors are expressed on astrocytes, we hypothesized that glucocorticoids regulate IL-1 receptors expression. IL-1beta-activated mouse primary astrocytes were treated with 10(-6) M dexamethasone, and IL-1 receptors were studied at the mRNA and protein levels. Using RT-PCR, IL-1RI and IL-1RII but not IL-1RAcP mRNAs were found to be up-regulated by dexamethasone in a time-dependent manner. Dexamethasone (Dex), but not progesterone, had no effect on IL-1RI but strongly increased IL-1RII mRNA expression. Binding studies revealed an increase in the number of IL-1RII binding sites under the effect of Dex, but no change in affinity. These findings support the concept that glucocorticoids have important regulatory effect on the response of astrocytes to IL-1.     

Corticosteroid induction of Ig+Ia- B cells in vitro is mediated via interaction with the glucocorticoid cytoplasmic receptor

Flow microfluorometry was used to examine the effect of dexamethasone on the expression of surface Ia (sIa) on resting and activated murine B cells. Although dexamethasone resulted in a 50% reduction in sIa expression 12 h after injection, it was significantly less suppressive when injected together with B cell activators. In vitro dexamethasone, but not other related steroid hormones, induced a population of cells that were sIg+sIa-. A 20% reduction in the expression of sIa was noted by 4 h of culture with 10 nM dexamethasone, but maximal inhibition of 70% was not reached until 12 h of culture, and this degree of suppression persisted as long as dexamethasone remained in culture. When the dexamethasone was washed out after 8 h of culture, the maximal reduction was still noted at 12 h, but by 24 h there was re-expression of sIa toward base line levels, indicating it did not induce irreversible lethal alterations in the B cell. The inhibition of sIa expression correlated with a specific reduction in the quantity of messenger RNA for sIa as measured by Northern blot analysis, indicating that this is mediated at least in part by suppression of the steady state levels of Ia mRNA. The corticosteroid receptor antagonist RU486 was able to reverse the suppressive effects of dexamethasone on sIa expression, thus demonstrating that its effect is mediated specifically by binding to its intracellular receptor. Furthermore, when protein synthesis was inhibited during the short period of time that cells were preincubated with dexamethasone, minimal suppression of Ia expression was noted, suggesting that the dexamethasone may be stimulating a protein that has suppressive effects on MHC class II expression. The suppressive effects of dexamethasone in vitro were substantially reduced when B cells were simultaneously activated by stimuli that increase the expression of sIa. These data indicate that the suppressive effects of corticosteroids on immune response Ag are corticosteroid specific; are greater in resting than in activated B cells; are induced via the classical steroid mechanism of action, which is receptor mediated; and may result from the induction of an inhibitory protein that suppresses Ia mRNA.     

Modulation of the phosphoinositide 3-kinase pathway alters innate resistance to polymicrobial sepsis

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-beta, IL-2, IL-6, IL-10, IL-12, and TNF-alpha in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.     

Cytokine production by dendritic cells genetically engineered to express IL-4: induction of Th2 responses and differential regulation of IL-12 and IL-23 synthesis

Dendritic cells (DCs) retrovirally transduced with IL-4 have recently been shown to inhibit murine collagen-induced arthritis and associated Th1 immune responses in vivo, but the mechanisms that underly these effects are not yet understood. In this report we demonstrate that IL-4-transduced DCs loaded with antigen led to lower T cell production of IFN-gamma, increased production of IL-4, and an attenuated, delayed type hypersensitivity response. We hypothesized that the ability of such DCs to regulate the Th1 immune response in vivo depends in part on their capacity to produce IL-12 and IL-23. Quantitative mRNA analysis revealed that IL-4-transduced DCs stimulated with CD40 ligand expressed higher levels of IL-12p35 mRNA, but lower levels of mRNA for IL-23p19 and the common subunit p40 found in both IL-12 and IL-23, compared with control DCs. These results, which indicate that expression of the IL-12 and IL-23 subunits is differentially regulated in IL-4-transduced DCs, were confirmed by ELISA of the IL-12 and IL-23 heterodimers. Thus, therapeutic suppression of Th1 -mediated autoimmunity (as recently shown in murine collagen-induced arthritis) and induction of Th2 responses in vivo by IL-4-transduced DCs occurs despite their potential to produce increased levels of IL-12, but could reflect, in part, decreased production of IL-23.     

UVB irradiation suppresses cytokine production and innate cellular immune functions in mice

We examined whether ultraviolet-B (UVB) irradiation (6 kJ/m2) alters cytokine production and other innate immune reactions by murine peritoneal macrophages and peripheral neutrophils. Along with these experiments, serum IgG levels were also assessed. In addition, using scanning electron microscopy (SEM) we observed macrophages that had been exposed to UVB in vitro. Results showed that UVB irradiation: (1) decreased IL-12 production while increasing IL-1alpha secretion from macrophages, but had no effect on IL-1alpha from neutrophils; (2) suppressed phagocytosis of macrophages but not of neutrophils; (3) diminished active oxygen production of macrophages but not of neutrophils; (4) had no effect on serum IgG levels; and (5) caused significant cell destruction of macrophages in vitro. These results suggested: (1) that UVB irradiation could induce characteristic suppression of innate immunity; (2) that innate cellular immunity was more susceptible to the effects of UVB irradiation than humoral immunity.     

KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection.

The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-gamma) secretion by murine spleen cells stimulated with KM(+), a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM(+), and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM(+) induced the secretion of IFN-gamma by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM(+). Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-gamma and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM(+) and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM(+) induces IL-12 p40 in vivo and has a protective effect against L. major infection.     

Transforming growth factor-β and Th17 responses in resistance to primary murine schistosomiasis mansoni

Discovery of the T-helper (Th) 17 cell lineage and functions in immune responses of mouse and man prompted us to investigate the role of transforming growth factor-beta (TGF-β) and interleukin (IL)-17 in innate resistance to murine schistosomiasis mansoni. Schistosoma mansoni-infected BALB/c and C57BL/6 mice were administered with recombinant TGF-β or mouse monoclonal antibody to TGF-β to evaluate the impact of this cytokine on host immune responses against lung-stage schistosomula, and subsequent effects on adult worm parameters. Developing schistosomula elicited increase in peripheral blood mononuclear cells (PBMC) mRNA expression and/or plasma levels of IL-4, IL-17, and interferon-gamma (IFN-γ), cytokines known to antagonize each other, resulting in impaired Th1/Th2, and Th17 immune responses and parasite evasion. Mice treated with TGF-β showed elevated PBMC mRNA expression of IL-6, IL-17, TGF-β, and TNF-α mRNA and increased IL-23 and IL-17 or TGF-β plasma levels, associated with significantly (P < 0.02–<0.0001) lower S. mansoni adult worm burden compared to controls in both mouse strains, thus suggesting that TGF-β led to heightened Th17 responses that mediated resistance to the infection. Mice treated with antibody to TGF-β showed increase in PBMC mRNA expression and plasma levels of IL-4, IL-12p70, and IFN-γ, and significantly (P < 0.02 and <0.0001) reduced worm burden and liver worm egg counts than untreated mice, indicating that Th1/Th2 immune responses were potentiated, resulting in significant innate resistance to schistosomiasis. The implications of these observations for schistosome immune evasion and vaccination were discussed.     

Recombinant human interleukin-1 inhibits the induction by dexamethasone of alkaline phosphatase activity in murine capillary endothelial cells

A mutual antagonism exists between interleukin-1s (IL-1s) as pro-inflammatory and glucocorticoids as anti-inflammatory mediators. This report examines the effects of IL-1 on the induction by dexamethasone of alkaline phosphatase in LEII murine endothelial cells. Dexamethasone increases the specific activity of alkaline phosphatase in a time- and dose-dependent fashion (maximum 14-fold induction at 10(-6) M, IC50 = 10(-8) M), and this induction can be completely inhibited by simultaneous incubation with picomolar concentrations of recombinant human IL-1 alpha or IL-1 beta. This IL-1-mediated antagonism of dexamethasone activity is not due to a down-regulation of glucocorticoid receptors in the cell line used, because the number of receptors and their affinity for dexamethasone is unchanged in IL-1-treated cells. However, induction of alkaline phosphatase by dexamethasone in LEII cells is receptor-mediated, since it can also be inhibited by glucocorticoid-receptor antagonists.     

Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.     

A cell-free extract of Salmonella typhimurium inhibits mitogen-induced proliferation of murine splenic T lymphocytes

Abstract In this study, a cell-free extract of Salmonella inhibited T cell mitogen-induced proliferation of spleen cells from non-immunized mice. The proliferation of murine spleen cells stimulated with a T cell mitogen, such as phytohaemagglutinin (PHA) or concanavalin A (ConA) was suppressed significantly when the cells were treated with a sonicate of S. typhimurium , but not of E. coli . The agent(s) responsible for the suppressive effect existed mainly in the soluble fraction of S. typhimurium , whereas the membrane fraction possessed minimal activity. The T cell proliferation suppression paralleled the level of interleukin-2 (IL-2) secretion. Addition of phorbol 12-myristate-13 acetate (PMA) to the cultures restored IL-2 secretion to normal levels, although proliferation remained suppressed and was not reversed by treatment with recombinant IL-2. These results suggest that the suppression of T cell proliferation induced by a soluble Salmonella fraction is associated with inhibition of IL-2 secretion and the response of T cells to IL-2 and the former effect is dependent upon the inhibition of the stimulatory activity of protein kinase C on IL-2 secretion. This type of suppression may explain a mechanism of immunosuppression induced by murine typhoid fever.     

Regulation of angiogenin expression in human HepG2 hepatoma cells by mediators of the acute-phase response.

Angiogenin is a potent inducer of neovascularization in vivo. However, like other angiogenic molecules, its specific physiologic roles and mechanisms regulating its expression remain to be elucidated. Angiogenin is a liver-derived component of normal serum whose concentration can increase in various disease states. This suggests that it might participate in the acute-phase response. In an initial study we showed that angiogenin protein and mRNA levels transiently increased in mice following an acute inflammatory stimulus. We now report that IL-6, a major inducer of acute-phase proteins, stimulates the synthesis and secretion of angiogenin protein in human HepG2 cells within 24 hr following treatment, an effect enhanced by dexamethasone. IL-6 also increases the amount of angiogenin mRNA without altering its half-life. This increase, suppressible by cycloheximide, peaks at 12 hr following stimulation and returns to basal levels by 48 hr. IL-1 alone slightly decreases the basal production of angiogenin protein and mRNA, but essentially abolishes the response to IL-6 in the absence or presence of dexamethasone. This antagonistic effect by IL-1 on IL-6 activity is not a result of changes in mRNA stability nor is it dependent on new protein synthesis. Thus, the combined effects of IL-6, IL-1, glucocorticoids, and perhaps other related factors may specifically control angiogenin expression. Since angiogenin is regulated in a manner similar to that of acute phase proteins both in vitro and in vivo, it may play a role in the host response to injury.