Effect of low-dose corticosterone pretreatment on the production of inflammatory mediators in super-low-dose LPS-primed immune cells.

Pretreatment of super-low-dose lipopolysaccharide (SL-LPS) induces a more hyperresponsive state on the production of proinflammatory mediators to a subsequent secondary challenge with high-dose LPS in innate immune cells. Low-dose glucocorticoids (GCs) are also known to induce inflammation and immunosuppression in the immune cells. However, there is limited knowledge on whether preconditioning of low-dose GCs enhances inflammatory responses and dysregulates T lymphocyte responses to secondary LPS in SL-LPS-primed immune cells. In the present study, RAW 264.7 and EL4 cells were pretreated with SL-LPS (50 pg/ml) or low-dose corticosterone (CORT50: 50 ng/ml and CORT100: 100 ng/ml) in fresh complete medium once a day for 2-3 days, consecutively, and then cultured in fresh complete medium for 6 or 24 h in the presence or absence of LPS (1-10 µg/ml) or concanavalin A (Con A). The results demonstrated that the repeated pretreatment of CORT50 strongly enhanced production of IL-6, IL-10, TNF-α, and nitric oxide (NO) by RAW 264.7 cells in EP (SL-LPS-primed cells: endotoxin priming) in the absence of LPS compared to those in control (vehicle-pretreated cells), whereas CORT100 reduced production of TNF-α and IL-10. Further, the repeated pretreatment of CORT50 markedly enhanced LPS-induced production of IL-6, IL-10, TNF-α, PGE2, and NO by RAW 264.7 cells in EP compared to those in control, whereas CORT100 attenuated LPS-induced production of IL-6, IL-10, and NO. Moreover, the repeated pretreatments of CORT50 and CORT100 greatly attenuated the Con A-stimulated production of IFN-γ and IFN-γ/IL-10 and LPS-stimulated production of IL-10, IFN-γ, and IFN-γ/IL-10 by SL-LPS-primed EL4 cells (EP). These findings suggest that double preconditionings of low grade hypercortisolemia and metabolic endotoxemia may act as important risk factors for metabolic disorder and severe morbidity and mortality in septic shock via upregulated production of inflammatory mediators and immunosuppression of IFN-γ-mediated responses.

Pretreatment of Low-Dose and Super-Low-Dose LPS on the Production of In Vitro LPS-Induced Inflammatory Mediators.

Pretreatment of low-dose lipopolysaccharide (LPS) induces a hyporesponsive state to subsequent secondary challenge with high-dose LPS in innate immune cells, whereas super-low-dose LPS results in augmented expression of pro-inflammatory cytokines. However, little is known about the difference between super-low-dose and low-dose LPS pretreatments on immune cell-mediated inflammatory and hepatic acute-phase responses to secondary LPS. In the present study, RAW 264.7 cells, EL4 cells, and Hepa-1c1c7 cells were pretreated with super-low-dose LPS (SL-LPS: 50 pg/mL) or low-dose LPS (L-LPS: 50 ng/mL) in fresh complete medium once a day for 2~3 days and then cultured in fresh complete medium for 24 hr or 48 hr in the presence or absence of LPS (1~10 µg/mL) or concanavalin A (Con A). SL-LPS pretreatment strongly enhanced the LPS-induced production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, TNF-α/IL-10, prostaglandin E2 (PGE2), and nitric oxide (NO) by RAW 264.7 cells compared to the control, whereas L-LPS increased IL-6 and NO production only. SL-LPS strongly augmented the Con A-induced ratios of interferon (IFN)-γ/IL-10 in EL4 cells but decreased the LPS-induced ratios of IFN-γ/IL-10 compared to the control, while L-LPS decreased the Con A- and LPS-induced ratios of IFN-γ/IL-10. SL-LPS enhanced the LPS-induced production of IL-6 by Hepa1c1c-7 cells compared to the control, while L-LPS increased IL-6 but decreased IL-1ß and C reactive protein (CRP) levels. SL-LPS pretreatment strongly enhanced the LPS-induced production of TNF-α, IL-6, IL-10, PGE2, and NO in RAW 264.7 cells, and the IL-6, IL-1ß, and CRP levels in Hepa1c1c-7 cells, as well as the ratios of IFN-γ/IL-10 in LPS- and Con A-stimulated EL4 cells compared to L-LPS. These findings suggest that pre-conditioning of SL-LPS may contribute to the mortality to secondary infection in sepsis rather than pre-conditioning of L-LPS.

Ripe fruit of Rubus coreanus inhibits mast cell-mediated allergic inflammation.

In this study, we investigated the effect of a water extract of the ripe fruits of Rubus coreanus Miq. (Rosaceae) (RFRC) on mast cell-mediated allergic inflammation and studied the possible mechanism of action. Mast cell-mediated allergic disease is involved in many diseases such as anaphylaxis, rhinitis, asthma and atopic dermatitis. RFRC dose-dependently inhibited compound 48/80-induced systemic anaphylaxis and serum histamine release in mice. RFRC reduced the immunoglobulin E (IgE)-mediated local allergic reaction, passive cutaneous anaphylaxis. RFRC attenuated histamine release from rat peritoneal mast cells and human mast cells by the reduction of intracellular calcium. RFRC decreased the phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187 (PMACI)-stimulated expression and secretion of pro-inflammatory cytokines in human mast cells. The inhibitory effect of RFRC on cytokine production was nuclear factor (NF)-κB- and mitogen-activated protein kinase (MAPK)-dependent. In addition, RFRC suppressed the activation of caspase-1. Our findings provide evidence that RFRC inhibits mast cell-derived allergic inflammatory reactions, and for the involvement of calcium, NF-κB, MAPKs and caspase-1 in these effects. Furthermore, in vivo and in vitro anti-allergic inflammatory effects of RFRC provide affirmative proof of a possible therapeutic application of this agent in allergic inflammatory diseases.

Farnesyl protein transferase inhibitory components of Polygonum multiflorum.

The methanolic extract of the roots of Polygonum multiflorum (Polygonaceae) was found to show inhibitory activity towards farnesyl protein transferase (FPTase). Bioassay-guided fractionation of the methanolic extract resulted in the isolation of two anthraquinone glycosides, as inhibitors of FPTase. These compounds inhibited the FPTase activity in a dose-dependent manner.

Prostaglandin E2-mediated dysregulation of proinflammatory cytokine production in pristane-induced lupus mice.

Systemic lupus erythematosus (SLE) is characterized by inflammatory and dysregulatory immune responses including overactive B cells, overproduction of proinflammatory cytokines, and T cell hyperactivity. PGE(2) modulates a variety of immune processes at sites of inflammation, including production of inflammatory cytokines. However, the role of PGE(2) in dysregulatory inflammatory and immune responses in lupus remains unclear. We investigated whether PGE(2) mediates production of inflammatory cytokines in pristane-induced lupus BALB/c mice. Our results showed that levels of serum and BAL PGE(2) and LPS-stimulated production of PGE(2) by peritoneal macrophages were remarkably increased in pristane-induced lupus mice compared to healthy controls. Exogenous PGE(2) enhanced production of IL-6, IL-10, and NO but decreased TNF-alpha by macrophages and augmented IFN-gamma, IL-6, and IL-10 by splenocytes from pristane-induced lupus mice compared to healthy controls. Exogenous PGE(2) also enhanced production of IFN-gamma, IL-6, and IL-10 by thymocytes from pristane-induced lupus mice. Indomethacin (Indo), a PGE(2) synthesis inhibitor, greatly inhibited LPS-induced production of IL-6 and IL-10 by macrophages from pristane-induced lupus mice, while enhanced TNF-alpha. Indo remarkably inhibited Con A-increased production of IFN-gamma, IL-6, and IL-10 by splenocytes and thymocytes from pristane-induced lupus mice. Therefore, our findings suggest that endogenous PGE(2) may mediate dysregulation of production of proinflammatory cytokines, such as IL-6, IL-10, and IFN-gamma, and NO in pristane-induced lupus mice.

Effects of Prunella vulgaris on mast cell-mediated allergic reaction and inflammatory cytokine production.

In this study, we investigated the effect of aqueous extract of Prunella vulgaris (Labiatae; PVAE) on the mast cell-mediated allergy model. We found that PVAE (0.001-0.1 g/kg) dose dependently inhibited compound 48/80-induced systemic anaphylaxis and serum histamine release in mice. PVAE decreased the IgE-mediated local allergic reaction, passive cutaneous anaphylaxis. In addition, PVAE attenuated phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated TNF-alpha, IL-6, and IL-8 secretion in human mast cells. The inhibitory effect of PVAE on proinflammatory cytokines was nuclear factor-kappaB (NF-kappaB) dependent. PVAE suppressed PMA and A23187-induced NF-kappaB/DNA binding activity and NF-kappaB-dependent gene reporter assay. Our findings provide evidence that PVAE inhibits mast cell-derived immediate-type allergic reactions and involvement of proinflammatory cytokines and NF-kappaB in these effects.

Immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the in vitro immune response in pristane-induced lupus mice.

Systemic lupus erythematosus (SLE) is characterized by overactive B cells that differentiate into autoantibody-forming cells, aberrant T cell function that provides helping B cells produce autoantibodies, and overproduction of proinflammatory cytokines. However, immunodysregulation in lupus pathogenensis remains incomplete. We examined mitogen-stimulated production of proinflammatory cytokines, cell proliferation, T cell activation, and T cell apoptosis in vitro in pristane-induced lupus BALB/c mice compared to normal mice. LPS-stimulated production of IL-6 and IL-10 by splenocytes and macrophages from pristane-induced lupus mice were remarkably up-regulated compared to normal mice, whereas production of macrophage TNF-alpha was significantly down-regulated. Moreover, in vitro production of IL-2, IL-6, IL-10 and IFN-gamma by Con A-stimulated splenocytes, cell proliferation in LPS- or Con A-stimulated- thymocytes and splenocytes, and expression of CD69+CD4+ T cells in Con A-stimulated splenocytes were greatly increased in cells derived from pristane-induced lupus mice compared to normal mice. In addition, splenic T cells and CD4+ T cells in thymocytes from pristane-induced lupus mice were more resistant than nonautoimmune normal cells to Con A-induced apoptosis. Our findings indicate that immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the in vitro immune responses in pristane-induced lupus mice may explain some of lupus pathogenesis.

Endotoxin induces late increase in the production of pulmonary proinflammatory cytokines in murine lupus-like pristane-primed model [corrected].

Lupus-like syndrome is characterized by multiple organ injuries including lungs and kidneys. Endotoxin induces a transiently intent systemic inflammatory response and indirectly transient acute lung injury in normal condition. However, whether endotoxin may trigger the persistent development of lung injury in chronic, inflammatory lupus-like syndrome compared with normal condition remains unclear. We examined the pulmonary vascular permeability and production of proinflammatory cytokines, such as TNF-alpha, IL-6, IL-10 and IFN-gamma, which play prominent roles in the pathogenesis of lupus-like tissue injury, 6 h and 72 h after i.p. lipopolysaccharide (LPS; endotoxin) injection in pristane-primed chronic inflammation ICR mice characterized by a lupus-like syndrome. These results demonstrated that levels of serum IL-6, IL-10 and IFN-gamma and bronchoalveolar lavage (BAL) IL-6 and IFN-gamma were remarkably increased 6 h in LPS-exposed pristane-primed mice compared with pristane-primed controls, while pulmonary vascular permeability and levels of serum and BAL TNF-alpha were not. And levels of BAL TNF-alpha, IL-6 and IL-10 were significantly enhanced 72 h in LPS-exposed pristane-primed mice compared with pristane-primed controls. Also, LPS significantly induced the increased in vitro production of TNF-alpha, IL-6 and IL-10 by lung cells obtained from LPS-exposed pristane-primed mice compared with LPS-exposed normal mice. Our findings indicate that LPS may trigger persistent progression of lung injury through late overproduction of BAL TNF-alpha, IL-6, and IL-10 in lupus-like chronic inflammation syndrome compared with normal condition.

The anti-anaphylactic effect of the gall of Rhus javanica is mediated through inhibition of histamine release and inflammatory cytokine secretion.

The immediate-type allergic reaction (anaphylaxis) is involved in many allergic diseases such as asthma, allergic rhinitis and sinusitis. We investigated the effect of the gall of Rhus javanica (GRJ) on the model of the immediate-type allergic reaction, and studied its possible mechanisms. GRJ inhibited compound 48/80-induced systemic reactions in mice. GRJ attenuated immunoglobulin (Ig) E-mediated local allergic reactions. In addition, GRJ dose dependently decreased histamine release from rat peritoneal mast cells activated by compound 48/80 or IgE. The decreasing effect of GRJ on the histamine release was mediated by the modulation of cAMP and [Ca2+]i in mast cells. Furthermore, GRJ decreased the phorbol 12-myristate 13-acetate plus calcium ionophore A23187-stimulated TNF-alpha and IL-6 secretion in human mast cells. The inhibitory effect of GRJ on the pro-inflammatory cytokine was c-Jun N-terminal kinase and nuclear factor-kappaB dependent. Our findings provide evidence that GRJ inhibits mast cell-derived immediate-type allergic reactions, and suggest the possible mechanisms of action.

Comparative study of the endotoxemia and endotoxin tolerance on the production of Th cytokines and macrophage interleukin-6: differential regulation of indomethacin.

Endotoxin tolerance reduces the capacity of monocytes to produce proinflammatory cytokines, results in cellular immune paralysis, and down-regulates the production of helper T (Th)1 type cytokines with a shift toward a Th2 cytokine response. Prostaglandin (PG)E2 in the immune system also results in macrophage inactivation and the suppression of Th1 activation and the enhancement of Th2 activation. However, the inhibitory effects of PGE2 on the altered polarization of the Th cell and macrophage interleukin (IL)-6 production characterized in part by cellular immune paralysis in a state of endotoxin tolerance is unclear. This study was undertaken, using indomethacin, to investigate the role of endogenous PGE2 on the Th cytokines and macrophage IL-6 production in a state of endotoxin tolerance compared to those with endotoxemia mice, wherein, in this latter case, the increased production of proinflammatory cytokines and PGE2 is exhibited. Endotoxemia was induced by injection of lipopolysaccharide (LPS; 10 mg/kg in saline) ip. once in BALB/c mice, and endotoxin tolerance was induced by pretreatment with LPS (1 mg/kg in saline) injected i.p. daily for two consecutive days and then with LPS 10 mg/kg on day 4. Splenocytes or macrophages were obtained from endotoxemia and endotoxin tolerance models pretreated with indomethacin, and then cytokine production was induced by Con A-stimulated splenocytes for the Th cytokine assays and LPS-stimulated macrophages for the IL-6 assay. Our results showed that endotoxemia led to significantly reduced IL-2 and IL-4 production, to significantly increased IL-6 production, whereas interferon (IFN)-gamma production was not affected. Indomethacin in the case of endotoxemia markedly attenuated IFN-gamma and IL-6 production and didnt reverse IL-2 and IL-4 production. Endotoxin tolerance resulted in the significantly reduced production of IL-2 and IFN-gamma and the significantly increased production of IL-4 and IL-6. Indomethacin in endotoxin tolerance greatly augmented IL-2 production, significantly decreased IL-4 production, and slightly attenuated IL-6 production. These findings indicate that endogenous PGE2 may mediate the suppressed Th1 type immune response, with a shift toward a Th2 cytokine response in a state of endotoxin tolerance, whereas endotoxemia may be regulated differentially. Also, endogenous PGE2 may mediate macrophage IL-6 production in the case of endotoxemia to a greater extent than in the case of endotoxin tolerance.