Viral hemorrhagic septicemia virus alters turbot Scophthalmus maximus macrophage nitric oxide production.

The effect of viral hemorrhagic septicemia virus (VHSV) in vitro infection on the nitric oxide (NO) production by turbot Scophthalmus maximus kidney macrophages has been addressed in the past. Previously, we had determined that only a small fraction of turbot possess head kidney macrophages that respond to a single exposure of lipopolysaccharide (LPS) with NO production (LPS-responsive macrophages), whereas macrophage cultures from other individuals were not activated by LPS alone and needed a combination of stimuli to respond (LPS-non-responsive macrophages). In the current work, we examined the effect of VHSV on NO production by macrophages characterized as LPS-responsive macrophages or LPS-non-responsive macrophages. Combinations of LPS and tumor necrosis factor alpha (TNF-alpha) and macrophage-activating factor (MAF) were also used to stimulate the cells for NO production. The effect of VHSV on NO production depends on the response to LPS alone. When a low multiplicity of infection was used (1.78 x 10(-3)), the NO production in response to LPS in LPS-responsive macrophages was significantly decreased. However, LPS-non-responsive macrophage cultures produced NO when a combination of LPS and VHSV was used. In the case of a higher VHSV multiplicity of infection (1.78), no significant change was observed in LPS-non-responsive animals. Combinations of LPS with TNF-alpha, LPS with MAF, and TNF-alpha with MAF were used to induce NO production in LPS-non-responsive macrophages. In all these cases, VHSV suppressed NO production, although at a significant level only when a combination of TNF-alpha and MAF was used for the induction of NO.     

CD40 ligation activates murine macrophages via an IFN-gamma-dependent mechanism resulting in tumor cell destruction in vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-gamma because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-gamma mAb, as well as in IFN-gamma-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-gamma for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-gamma. We confirmed this hypothesis by detecting intracytoplasmic IFN-gamma in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-gamma production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-gamma and mediate tumor cell destruction.     

Comparative effect of ion calcium and magnesium in the activation and infection of the murine macrophage by Leishmania major

Amastigotes of Leishmania major have a great ability to evade destruction in host cells. This study investigated the activation in resident, inflammatory macrophages and J774 cells in vitro treated with lipopolysaccharide (LPS), soluble Leishmania antigen (SLA), calcium ionophore (CaI) and magnesium (Mg2+) alone or combined. An increase in nitric oxide (NO) production was observed in J774 or inflammatory macrophages treated with LPS alone or in combination with SLA and CaI. The same treatments did not affect the NO release by resident macrophages. There was no interference in uptake of L. major but CaI decreased intracellular proliferation of the parasite. This study demonstrated the importance of CaI in decreasing L. major proliferation inside murine macrophages while Mg2+ seemed to increase parasite proliferation. These finding may help to understand the events involved in host cells' clearance of this pathogen.     

The in vitro anti-platelet,antioxidant and cellular immunity activity of <Emphasis Type="Italic">Phellinus gilvus</Emphasis> fractional extracts

The in vitro anti-platelet and antioxidant activities of various solvent extracts from Phellinus gilvus (PG), and the effects of hot water extract from PG (PGW) on murine cellular immunity were investigated. Chloroform extract (CE), methanol extract (ME) and butanol extract (BE) from PG could significantly inhibit platelet aggregation induced by thrombin. Ethyl acetate extract (EAE), BE, ME from PG had significant 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity compared with the control, and the EAE showed the highest effect with IC₅₀ values of 13.34 μg/ml, which is higher than that of ascorbic acid (40 μg/ml). In addition, EAE displayed the inhibition of xanthine oxidase (XO) activity with IC₅₀ value of 2.45 μg/ml. As to the cellular immunity activity, PGW could enhance both the lipopolysaccharide (LPS)-induced B lymphocyte proliferation and concanavalin A (Con A)-induced T lymphocyte proliferation in vitro. The phagocytosis of both peritoneal macrophages and RAW264.7 macrophage cells were also increased by the addition of PGW. Moreover, PGW was found to inhibit the nitric oxide (NO) production of RAW264.7 macrophages induced by LPS in a concentration-dependant manner.     

TNF-alpha, H2O2 and NO response of peritoneal macrophages to Yersinia enterocolitica O:3 derivatives

In this study, the effect of Yersinia derivatives on nitric oxide (NO), hydrogen peroxide (H2O2) and tumor necrosis factor-alpha (TNF-alpha) production by murine peritoneal macrophages was investigated. Addition of lipopolysaccharide (LPS) to the macrophage culture resulted in NO production that was dose dependent. On the other hand, bacterial cellular extract (CE) and Yersinia outer proteins (Yops) had no effect on NO production. The possible inhibitory effect of Yops on macrophage cultures stimulated with LPS was investigated. Yops partially inhibited NO production (67.4%) when compared with aminoguanidine. The effects of Yersinia derivatives on H2O2 production by macrophages were similar to those on NO production. LPS was the only derivative that stimulated H2O2 release in a dose-dependent manner. All Yersinia derivatives provoked the production of TNF-alpha, but LPS had the strongest effect, as observed for NO production. CE and Yops stimulated TNF-alpha production to a lesser extent than LPS. The results indicate the possibility that in vivo Yops may aid the evasion of the bacteria from the host defense mechanism by impairing the secretion of NO by macrophages.     

In vitro augmentation of rat natural killer (NK) cell activity

In vitro augmentation of rat natural killer (NK) cell activity was produced by 2 types of treatment. Increased activity occurred "spontaneously" when spleen cells were cultured alone at 37 degrees C. This augmentation was dependent on the presence of adherent, phagocytic cells, presumably macrophages, and was independent of LPS of FCS. Normally low levels of NK activity, present in macrophage-depleted cultured cells, could also be boosted in vitro by incubation with Corynebacterium parvum. This augmentation appeared to be independent of both B cells and macrophages and may be due to stimulation of rat NK cells themselves. Both forms of augmentation were associated with the production of interferon, were found in rats of all ages and strains tested, and should provide an excellent in vitro system for detailed studies of activation of rat NK cells.     

Early-appearing tumor-infiltrating natural killer cells play an important role in the nitric oxide production of tumor-associated macrophages through their interferon production

 Both natural killer (NK) cells and macrophages are thought to be the main effectors responsible for early antitumor defense. In this study, we investigated the role of tumor-infiltrating NK cells in initiating nitric oxide (NO) production by tumor-associated macrophages (TAM). The in vivo depletion of NK cells prior to the i.p. inoculation of melanoma cells resulted in a significant decrease in the NO production of the TAM prepared from the peritoneal exudate cells (PEC). Such prior NK cell depletion also decreased the ability of TAM to show any antitumor activity in vitro. The addition of N ^G-monomethyl-L-arginine (Me-L-Arg) to the culture partially inhibited the ability of TAM to suppress the proliferation of melanoma cells and also decreased their cytolytic activity against melanoma cells. These results suggest that the TAM exhibited both cytostatic and cytolytic activities through their NO production. In an in vivo assay, the administration of Me-L-Arg permitted the more rapid growth of i.p. inoculated melanoma cells compared with the control. On the other hand, the decreased NO production of TAM, resulting from the prior NK cell depletion, was restored by the i.p. administration of interferon γ (IFNγ). In addition, the in vivo administration of anti-IFNγ mAb into mice inoculated i.p. with melanoma cells also significantly decreased the NO production of TAM in peritoneal exudate cells. Furthermore, the tumor-infiltrating NK cells produced a considerable level of IFNγ. Overall, these results indicate that early-appearing tumor-infiltrating NK cells play an important role in the NO production of TAM through their IFNγ production. Received: 11 March 1997 / Accepted: 31 July 1997     

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.     

The effect of vaccination with the lysate of heat-shocked tumor cells on nitric oxide production in BALB/c mice with fibrosarcoma tumor

The aim of this study was to investigate the effect of heat shock protein-70 (HSP-70) on splenocyte proliferation and nitric oxide (NO) production in the BALB/c mice fibrosarcoma tumor model. To do so, HSP-70 was induced in the lysate of heat-shocked tumor cells and WEHI-164 cells (mouse fibrosarcoma cell line) were injected subcutaneously into the right flank of inbred BALB/c mice to establish a tumor model. Three animal bearing tumor groups were applied: the test group; vaccinated with HSP-70 enriched tumor lysate; control group I, vaccinated with tumor lysate only; and control group II, which received PBS. Using immunoblot analysis, an increase of HSP-70 expression was detected in the lysate of heat-shocked cells in comparison with non-heat-shocked cells. The effect of the test lysate on NO production was measured both in vitro and in vivo in the peritoneal macrophages and splenocytes of tumor bearing mice, respectively. The result showed a significant increase in NO production both in vitro by peritoneal macrophages and in vivo after immunization with HSP-70 enriched tumor lysate. In addition, tumor growth was significantly postponed and the proliferation of splenocytes was increased in the test group. Our results indicate that the lysate of heat-shocked tumor cells was more potent than that of non-heat-shocked tumor cells in inducing anti-tumor immunity. Since production of NO by HSP-activated antigen presenting cells (APCs) is likely to affect innate immunity and tumor growth, the probable mechanism of postponing tumor growth would be NO production by innate immune cells. These findings provide a useful therapeutic model for developing novel approaches to cancer treatments.     

The Immunoproteasomes Regulate LPS-Induced TRIF/TRAM Signaling Pathway in Murine Macrophages

We have proposed the novel concept that the macrophage ubiquitin-proteasome pathway functions as a key regulator of Lipopolysaccharide (LPS)-induced inflammation signaling. These findings suggest that proteasome-associated protease subunits X, Y, and Z are replaced by LMP subunits after LPS treatment of RAW 264.7 cells. The objective here was to determine the contribution of selective LMP proteasomal subunits to LPS-induced nitric oxide (NO) and TNF-α production in primary murine macrophages. Accordingly, thioglycollate-elicited macrophages from LMP7, LMP2, LMP10 (MECL-1), and LMP7/MECL-1 double knockout mice were stimulated in vitro with LPS, and were found to generate markedly reduced NO levels compared to wild-type (WT) mice, whereas TNF-α levels responses were essentially unaltered relative to wild-type responses. The recent studies suggest that the TRIF/TRAM pathway is defective in LMP knockouts which may explain why iNOS/NO are not robustly induced in LPS-treated macrophages from knockouts. Treating these macrophages with IFN-γ and LPS, however, reverses this defect, leading to robust NO induction. TNF-α is induced by LPS in the LMP knockout macrophages because IκB and IRAK are degraded normally via the MyD88 pathway. Collectively, these findings strongly support the concept that LMP7/MECL-1 proteasomes subunits actively function to regulate LPS-induced NO production by affecting the TRIF/TRAM pathway.     

Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxin-induced nitric oxide

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-beta prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-alpha plus IFN-gamma are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.     

Tumor-associated myeloid cells can be activated in vitro and in vivo to mediate antitumor effects

Tumor growth is often accompanied by the accumulation of myeloid cells in the tumors and lymphoid organs. These cells can suppress T cell immunity, thereby posing an obstacle to T cell-targeted cancer immunotherapy. In this study, we tested the possibility of activating tumor-associated myeloid cells to mediate antitumor effects. Using the peritoneal model of B16 melanoma, we show that peritoneal cells (PEC) in tumor-bearing mice (TBM) had reduced ability to secrete nitric oxide (NO) following in vitro stimulation with interferon gamma and lipopolysaccharide, as compared to PEC from control mice. This reduced function of PEC was accompanied by the influx of CD11b(+) Gr-1(+) myeloid cells to the peritoneal cavity. Nonadherent PEC were responsible for most of the NO production in TBM, whereas in na?ve mice NO was mainly secreted by adherent CD11b(+) F4/80(+) macrophages. Sorted CD11b(+) Gr-1(-) monocytic and CD11b(+) Gr-1(+) granulocytic PEC from TBM had a reduced ability to secrete NO following in vitro stimulation (compared to na?ve PEC), but effectively suppressed proliferation of tumor cells in vitro. In vivo, treatment of mice bearing established peritoneal B16 tumors with anti-CD40 and CpG resulted in activation of tumor-associated PEC, reduction in local tumor burden and prolongation of mouse survival. Inhibition of NO did not abrogate the antitumor effects of stimulated myeloid cells. Taken together, the results indicate that in tumor-bearing hosts, tumor-associated myeloid cells can be activated to mediate antitumor effects.     

Mass separation and in vitro immunological activity of membrane-fractionated polysaccharides from fruiting body and mycelium of Agaricus subrufescens

Membrane technology has been applied to separate polysaccharides from Agaricus subrufescens (ASPs). The membrane-retained fractions and unfractionated preparations have been tested for in vitro immunological activity. Both the microfiltration (MF) and ultrafiltration (UF1) membranes were able to separate high-molecular weight polysaccharides from fruiting body (ASP-FB) and submerge-fermented mycelium (ASP-SmF) extracts. All fractions showed immunostimulatory effects on RAW 264.7 macrophages, measured by TNF-??, iNOs gene expression, and NO production. In contrast, antibody and proliferation levels in B lymphoblastoid SKW 6.4 cells were significantly increased after treatment with ASP-FB, but did not with ASP-SmF preparations. The ASPs- and LPS-induced stimulation could be differentiated by the finding that polymyxin B, a specific inhibitor of LPS, did not significantly affect the immunoactivating response and proliferation activity of ASPs on macrophages and B cells, respectively. Furthermore, the ASP-FB treatment was unable to induce IL-6 production by B cells unlike LPS activation, sustaining distinct signaling pathways for ASP-FB and LPS. The overall results provided additional information about the action of ASPs on the immune system and support the membrane method to separate and concentrate high-molecular weight ASPs for immunopharmacological and biotechnological applications.     

Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction

Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII). We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM). The addition of the NO donor DETA NONOate (200 microM) inhibited BMM proliferation by approx. 80%. However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII-/- mice as from wild-type mice. Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea. Moreover, in both normal and NOSII-/- BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types. Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression. Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.     

Nitric oxide production by murine spleen cells stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could induce murine spleen cells to produce nitric oxide (NO). Spleen cells derived from Balb/c mice were stimulated with LPS-A. actinomycetemcomitans or LPS from Escherichia coli for 4 days. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B, and cytokines (IFN-gamma and IL-4) on the production of NO were also assessed. The NO production from the carrageenan-treated spleen cells stimulated with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma was determined. The carrageenan-treated mice were transferred with splenic macrophages and the NO production was assessed from the spleen cells stimulated with LPS-A. actinomycetemcomitans or LPS-A. actinomycetemcomitans and IFN-gamma. The results showed that NO production was detectable in the cultures of spleen cells stimulated with LPS-A. actinomycetemcomitans in a dose-dependent fashion, but was lower than in the cells stimulated with LPS from E. coli. The NO production was blocked by NMMA and polymyxin B. IFN-gamma up-regulated but IL-4 suppressed the production of NO by the spleen cells stimulated with LPS-A. actinomycetemcomitans. The carrageenan-treated spleen cells failed to produce NO after stimulation with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma. Adoptive transfer of splenic macrophages to the carrageenan-treated mice could restore the ability of the spleen cells to produce NO. The results of the present study suggest that LPS-A. actinomycetemcomitans under the regulatory control of cytokines induces murine spleen cells to produce NO and that splenic macrophages are the cellular source of the NO production. Therefore, these results may support the view that NO production by LPS-A. actinomycetemcomitans-stimulated macrophages may play a role in the course of periodontal diseases.     

Priming and activation of mouse macrophages by trehalose 6,6'-dicorynomycolate vesicles from Corynebacterium glutamicum

Vesicles consisting of pure trehalose dicorynomycolate (TDCM), the corynebacterial analog of the most studied mycobacterial glycolipid 'cord factor', were isolated from Corynebacterium glutamicum cells by mild detergent treatment; these induced in vivo a macrophage priming similar to that obtained with mycobacterial-derived trehalose dimycolate. In vitro, both TDCM and bacterial lipopolysaccharide (LPS) induced in macrophages the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), endotoxin tolerance, and were primed for an enhanced secondary NO response to LPS. Interferon-gamma pretreatment did not influence the LPS-induced TNF-alpha response, but considerably increased the TDCM-induced response.     

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.