Splenocyte IFN-gamma Production Research Articles

Potentiating effects of high-molecular weight fucoidan-agaricus mix (CUA) feeding on tumor vaccination

Abstract Fucoidan is a series of sulfated polysaccharides derived from brown algae that mainly consist of L-fucose. Various biological activities of fucoidan such as anti-cancer and immune modulatory effects were reported, and our investigation with animal experiments and human trials demonstrated fucoidan from Cladosiphon okamuranus and Undaria pinnatifida effectively augmented anti-tumor immunity in combination with Agaricus blazei mycelium extract. In this study, we evaluated dietary effects of the two types of high-molecular weight fucoidans and the agaricus extract mix (CUA) on achievement of effective tumor vaccination with a tumor antigen gp70 expressed on colon-26 tumor cell line. Balb/c mice were immunized with 0.05 mg gp70 peptide emulsified in complete freund’s adjuvant and intake 1% CUA containing AIN93G diet for 4 weeks. This procedure totally enhanced systemic immune function because splenocytes from the vaccinated mice extensively proliferate in response to concanavalin A-stimulation. The NK cell activity and gp70 peptide-stimulated IFN-gamma production in splenocytes from the vaccinated mice were tended to augment by the CUA feeding. On the other hands, the CUA feeding potentiated the killing activity to colon-26 carcinoma of draining lymph node (LN) cells from the vaccinated mice in association with increase of gp70-specific CD8-positive T cell population. Furthermore, the expressions of MHC class II molecule (I-A/I-E) on CD11c-positive and F4/80-positive populations of LN cells from the vaccinated mice were elevated by the intake of CUA. These results suggested that the CUA feeding potentially support effective induction of anti-tumor immune function by vaccination with tumor antigen peptides.

Leuconostoc citreumHJ-P4 (KACC 91035) regulates immunoglobulin E in an ovalbumin-induced allergy model and induces interleukin-12 through nuclear factor-kappa B and p38/c-Jun N-terminal kinases signaling in macrophages

Leuconostoc citreum (L. citreum) HJ-P4 (KACC 91035) is one of the major predominant species in kimchi fermentation in Korea. The purpose of the present study was to test the immunomodulatory capacity of L. citreum to modulate the IgE-mediated allergic response and to examine the involvement of NF-kappaB and MAPK in IL-12 production in macrophages. Balb/c mice were sensitized with OVA/alum and oral administration of L. citreum to the mice began before or after the OVA sensitization. Protein and mRNA expression of Th1 cytokines in splenocytes by L. citreum in vitro was measured. The role of NF-kappaB and MAPK such as p38, ERK1/2 and JNK in L. citreum-induced IL-12 was investigated in peritoneal macrophages and RAW264.7 cell lines. L. citreum inhibited the serum levels of total IgE, IgG1 and IgG2a altogether and increased OVA-specific IFN-gamma production in splenocytes from pre- and post-sensitized animals. However, the downregulation of IL-4 and IL-5 production was observed only in the pre-sensitization group. The ability of L. citreum to stimulate IFN-gamma was dependent on its induction of IL-12. NF-kappaB, p38 and JNK were mainly involved in L. citreum-induced IL-12 production. In conclusion, the current study demonstrated that L. citreum is able to regulate serum IgE generation at the induction and effector phases of allergic response through overall control over antibody production and that its involvement of IL-12 production was mediated through NF-kappaB and p38/JNK. Taken together, the use of L. citreum can be useful in preventing the development and progression of IgE production.

Antitumor and antimetastatic actions of anthrone‐C‐glucoside, cassialoin isolated from Cassia garrettiana heartwood in colon 26‐bearing mice

We examined the antitumor and antimetastatic actions of 10-hydroxy-anthrone-C-glucoside cassialoin isolated from Cassia garrettiana heartwood in colon 26-bearing mice. Cassialoin (5 and 10 mg/kg) inhibited tumor growth and metastasis to the abdomen and the expression of CD31 (angiogenesis marker) in the tumors, and it increased the numbers of the gamma-interferon (IFN-gamma)-positive, CD8(+) T and natural killer cells in the small intestine or spleen of colon 26-bearing mice. Furthermore, cassialoin inhibited tumor-induced angiogenesis in colon 26-packed chamber-bearing mice. We examined the metabolic activities in the blood, stomach and small intestine after p.o. administration of cassialoin to mice. These results suggest that cassialoin might be converted to chrysophanol through chrysophanol-9-anthrone and metabolized to aloe-emodin from chrysophanol. Chrysophanol-9-anthrone inhibited vascular endothelial growth factor (VEGF) and matrix metallopeptidase-9 expression in colon 26 cells at 5 and 10 microM, and it inhibited VEGF-induced angiogenesis and migration in human umbilical vein endothelial cells (HUVEC) at 0.5-10 microM. Furthermore, chrysophanol-9-anthrone inhibited VEGF receptor (VEGFR)-2 expression and VEGF-induced VEGFR-2 phosphorylation. Aloe-emodin also inhibited the VEGF-induced angiogenesis by HUVEC at 1-100 microM. Cassialoin, chrysophanol-9-anthrone and aloe-emodin enhanced concanavalin A-induced IFN-gamma production in splenocytes of colon 26-bearing mice at a low concentration of 0.1 microM. From these results, it is suggested that the antitumor and antimetastatic actions of p.o. administered cassialoin may be partly due to cassialoin and its metabolites such as chrysophanol-9-anthrone and aloe-emodin through their anti-angiogenic activities and/or the modulation of the immune systems in the spleen and small intestine in tumor-bearing mice.

Protein-energy malnutrition decreases immune response toLeishmania chagasivaccine in BALB/c mice

Protein-energy malnutrition and visceral leishmaniasis are important problems of public health affecting millions of people worldwide. Vaccine efficacy depends on the ability of individuals to mount an appropriate immune response and may be inadequate in malnourished persons. In this study, we used a mouse model to verify the effect of combined protein, iron and zinc deficiency in the response to Leishmania chagasi antigen vaccine. BALB/c mice were fed with a low-protein (3% casein), iron- and zinc-deficient diet or control diet (14% casein and sufficient in zinc and iron). After malnutrition establishment, mice were vaccinated subcutaneously with L. chagasi Ag plus saponin. After vaccination, mice were nutritionally repleted and then all mice were challenged with L. chagasi promastigotes. Four weeks later, liver and spleen parasite load was evaluated. Our data show that vaccine caused a significant reduction in parasite load in spleen and liver from mice fed with control diet. However, splenic parasitism was increased in mice fed with deficient diet and this diet caused a reduction in splenocyte IFN-gamma production in response to the vaccine in repleted mice. These data suggest that malnutrition may alter immune response to L. chagasi vaccine in BALB/c model of infection, even after nutritional repletion.

Toxoplasma gondii: expression and characterization of a recombinant protein containing SAG1 and GRA2 in Pichia pastoris

Toxoplasma gondii is an obligate intracellular protozoan which infects most species of warm-blooded animals and causes toxoplasmosis. Previous immunological and immunization studies have demonstrated the potential role of T. gondii antigens SAG1 and GRA2 as a vaccine candidate. In the present study, we have cloned, expressed, and purified a recombinant protein SAG1-GRA2 in Pichia pastoris. Results showed that P. pastoris was a robust system producing a large amount of highly purified and biological activity protein. BALB/c mice immunized with SAG1-GRA2 elicited stronger humoral and cellular responses in comparison to control groups. This immunization resulted in an enhanced Th1 immune response as measured by IgG2a antibody production and increased splenocyte IFN-gamma production, whereas no IL-4 was detected. After a lethal challenge with the highly virulent T. gondii RH strain, a prolonged survival time in SAG1-GRA2-immunized mice was observed in comparison to control groups. Our data demonstrate that SAG1-GRA2 triggered a protective response against toxoplasmosis. Therefore, SAG1-GRA2 protein might be a good candidate for the further development of a multiantigenic vaccine.

Absence of reuptake of serotonin influences susceptibility to clinical autoimmune disease and neuroantigen-specific interferon-gamma production in mouse EAE.

Serotonin (5-hydroxytryptamine, 5-HT) is one of the most extensively studied neurotransmitters of the central nervous system. It also has been identified in constituents of the immune system. Therefore serotonin has been suggested to serve as a mediator of bidirectional interactions between the nervous system and the immune system. We investigated this interaction in experimental autoimmune encephalomyelitis (EAE), a well-defined animal model of autoimmune disease of the central nervous system (CNS) mimicking features of the human disease multiple sclerosis. EAE was induced by immunization with the autoantigens myelin basic protein (MBP) or the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG) spanning amino acids 35-55 (MOGp 35-55). We studied EAE in knockout (KO) mice lacking the 5-HT transporter (5-HTT) on a C57.BL/6 background, in comparison with wild-type C57.BL/6 animals. After immunization with MOGp 35-55, or with rat MBP, the disease courses of the 5-HTT knockout mice were attenuated as compared to wildtype control mice. This difference was more pronounced in female animals. To dissect potential immune mechanisms underlying this phenomenon, histological studies of the CNS and cytokine measurements in mononuclear cells from the spleens of 5-HTT KO mice and wild-type controls were performed. We found a reduction of the inflammatory infiltrate in the CNS and of the neuroantigen-specific production of IFN-gamma in splenocytes, again accompanied by a gender difference. These findings suggest a potential role of extracellular 5-HT homeostasis in the fine-tuning of neuroantigen-specific immune responses.

The interleukin-1 beta-converting enzyme inhibitor pralnacasan reduces dextran sulfate sodium-induced murine colitis and T helper 1 T-cell activation.

The proinflammatory cytokines interleukin (IL)-1beta and IL-18 are supposed to play a crucial role in the pathogenesis of human inflammatory bowel disease. To exert biological activity, the precursors of both IL-1beta and IL-18 need to be cleaved by the interleukin-1beta-converting enzyme (ICE). IL-18 induces the synthesis of IFN-gamma in T cells and NK cells. In the present study, we investigated the effect of the specific ICE inhibitor pralnacasan in dextran sulfate sodium-induced murine colitis. Colitis was induced in BALB/c mice by 3.5% dextran sulfate sodium dissolved in drinking water for 10 days. Pralnacasan was administered either intraperitoneally or orally every day. To assess in vivo efficacy, a clinical disease activity score was evaluated daily. Colon length, expression of IL-18 in colonic tissue, expression of interferon-gamma (IFN-gamma) in paraaortal lymphocytes, and systemic production of IFN-gamma in splenocytes were analyzed post mortem. Intraperitoneally administered pralnacasan significantly reduced the clinical score compared with the dextran sulfate sodium control group from day 6 to day 10. Oral administration of pralnacasan also significantly reduced the clinical score at days 8 and 9. Administration of pralnacasan i.p. reduced the expression of intracolonic IL-18 significantly. Furthermore, pralnacasan reduced the number of IFN-gamma-positive lymphocytes in paraaortal lymph nodes. IFN-gamma synthesis in stimulated splenocytes was significantly suppressed in all pralnacasan-treated groups. No side effects of pralnacasan were observed. In conclusion, pralnacasan is effective in the prevention of dextran sulfate sodium-induced colitis. This effect is probably mediated by suppression of the proinflammatory cytokines IL-18, IL-1beta, and IFN-gamma.

Adoptive transfer of apoptotic splenocytes worsens survival, whereas adoptive transfer of necrotic splenocytes improves survival in sepsis.

In sepsis, both necrotic and apoptotic cell death can occur. Apoptotic cells induce anergy that could impair the host response, whereas necrotic cells cause immune activation that might result in enhanced antimicrobial defenses. We determined whether adoptive transfer of apoptotic or necrotic cells impacted survival in a clinically relevant sepsis model. We also evaluated the effects of adoptive transfer of apoptotic or necrotic cells on the prototypical TH1 and TH2 cytokines IFN-gamma and IL-4, respectively. C57BL6/J mice had adoptive transfer of apoptotic (irradiated) or necrotic (freeze thaw) splenocytes. Controls received saline. Apoptotic cells greatly increased mortality, whereas necrotic splenocytes markedly improved survival, P < or = 0.05. The contrasting effects that apoptotic or necrotic cells exerted on survival were mirrored by opposite effects on splenocyte IFN-gamma production with greatly decreased and increased production, respectively. Importantly, either administration of anti-IFN-gamma antibodies or use of IFN-gamma knockout mice prevented the survival benefit occurring with necrotic cells. This study demonstrates that the type of cell death impacts survival in a clinically relevant model and identifies a mechanism for the immune suppression that is a hallmark of sepsis. Necrotic cells (and likely apoptotic cells) exert their effects via modulation of IFN-gamma

Immune deviation away from Th1 in interferon-gamma knockout mice does not enhance TSH receptor antibody production after naked DNA vaccination.

TSH receptor (TSHR) DNA vaccination induces high TSHR antibody levels in BALB/c mice housed in a conventional facility. However, under pathogen-free conditions, we observed a Th1 cellular response to TSHR antigen characterized by interferon-gamma (IFN gamma) production. In the present study we investigated the effect on TSHR DNA vaccination of diverting the cytokine milieu away from Th1 using 1) IFN gamma knockout BALB/c mice, and 2) wild-type mice covaccinated with DNA for the TSHR and for IFN gamma/receptor-Fc protein that prevents IFN gamma from binding to its receptor. Neither approach enhanced TSHR antibody levels, although splenocyte IFN gamma production in response to TSHR antigen was absent (IFN gamma knockouts) or reduced (IFN gamma receptor-Fc). Moreover, production of IL-2, another Th1 cytokine, but not Th2 cytokines, indicated that neither strategy overcame the Th1 bias of im DNA vaccination. Importantly, splenocyte production of IFN gamma and IL-2 provides a sensitive detection system for TSHR-specific T cells. Unexpectedly, higher TSHR antibody levels developed in rare mice. High titer animals had TSHR-specific responses of both Th2 and Th1 types, whereas low titer animals had Th1-restricted TSHR responses. The heterogeneity of responses induced by TSHR DNA vaccination in mice may provide insight into the titers and IgG subclasses of spontaneous autoantibodies in humans.

Immunomodulatory effects of morphine withdrawal in the rat are time dependent and reversible by clonidine

It is well established that opioids can modulate the immune status following both acute and chronic administration and that tolerance develops to some of these immunomodulatory effects. Few studies, however, have investigated opioid withdrawal-induced immunomodulation and the mechanism by which that process may be mediated. The present study examines the immunomodulatory properties of morphine withdrawal alone and in the presence of the alpha(2)-adrenergic agonist, clonidine. Rats drank a morphine solution for 20 days; withdrawal was induced on day 21 by replacing the morphine solution with plain tap water. Measurements of withdrawal-induced weight change and immunomodulation were obtained at several time points after withdrawal induction. Immune status was assessed by determining concanavalin A (Con-A), toxic shock syndrome toxin (TSST-1), and lipopolysaccharide (LPS)-stimulated splenocyte proliferation, splenic ConA-stimulated interferon (IFN)-gamma production, and splenic natural-killer (NK) cell activity. In a separate series of experiments, systemic injections of clonidine (0.001-0.01 mg/kg) were administered during a 12-h withdrawal episode and all measures of immune status were reassessed. Weight change was time dependent, with peak decreases in weight occurring 24 h following withdrawal induction. Rats also exhibited a time-dependent suppression of immune status in all assays except LPS-stimulated proliferation; immunomodulation was most evident 12 h following withdrawal induction. Clonidine dose dependently prevented withdrawal-induced suppression of Con-A and TSST-1-stimulated splenocyte proliferation, Con-A-stimulated splenocyte IFN-gamma production, and splenic NK cell activity. These findings demonstrate that opioid withdrawal significantly suppresses a subset of immune parameters and that these effects can be prevented by clonidine.

Evidence for differential induction of helper T cell subsets during Trichinella spiralis infection.

The H-2-compatible mouse strains, AKR and B10.BR, exhibit disparate responses to infection with the parasitic nematode Trichinella spiralis. The resistant AKR mice expel intestinal adult worms faster than susceptible B10.BR mice. We tested antibody and lymphokine responses in these strains. With respect to antibody responses, the B10.BR mice had 3- to 10-fold more serum IgE and T. spiralis-specific IgG1 and IgA than AKR mice. The B10.BR mice also had greater numbers of IgG and IgA plaque-forming cells than AKR mice. In contrast, AKR mice produced T. spiralis-specific IgG2a, whereas the B10.BR mice did not. The antibody response kinetics of these strains were similar. We also analyzed lymphokine secretion after restimulating lymphocytes in vitro with T. spiralis Ag. The AKR mesenteric lymph node cells produced more IFN-gamma and less IL-4 than the B10.BR mesenteric lymph node cells. The B10.BR splenocytes produced more IL-4 than the AKR splenocytes, although splenocyte IFN-gamma production was not different. The kinetics of IL-4 production also differed between the two strains. In summary, resistant AKR mice produced more IFN-gamma and T. spiralis-specific IgG2a than susceptible B10.BR mice, which produced more IL-4, IgE, and T. spiralis-specific IgG1. Our results are consistent with differential activation of Th cell subsets in T. spiralis-infected AKR and B10.BR mice.