Kyu Hwan Yang

Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism

Cannabinoid compounds, including the major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (delta 9-THC), have been widely established as being inhibitory on a broad array of humoral and cell-mediated immune responses. The presence of cannabinoid receptors has been identified recently on mouse spleen cells, which possess structural and functional characteristics similar to those of the G-protein coupled cannabinoid receptor originally identified in rat brain. These findings, together with those demonstrating that delta 9-THC inhibits adenylate cyclase in splenocytes, strongly suggest that certain aspects of immune inhibition by cannabinoids may be mediated through a cannabinoid receptor-associated mechanism. The objective of the present studies was to determine whether inhibition of adenylate cyclase is relevant to mouse spleen cell immune function and, if so, whether this inhibition is mediated through a Gi-protein coupled mechanism as previously described in neuronal tissue. Spleen cell activation by the phorbol ester phorbol-12-myristate-13-acetate (PMA), plus the calcium ionophore ionomycin, produced a rapid but transient increase in cytosolic cAMP, which was inhibited completely by immunosuppressive concentrations of delta 9-THC (22 microM) and the synthetic bicyclic cannabinoid CP-55940 (5.2 microM), which produced no effect on cell viability. Inhibition by cannabinoids of lymphocyte proliferative responses to PMA plus ionomycin and sheep erythrocyte (sRBC) IgM antibody-forming cell (AFC) response, was abrogated completely by low concentrations of dibutyryl-cAMP (10-100 microM). Inhibition of the sRBC AFC response by both delta 9-THC (22 microM) and CP-55940 (5.2 microM) was also abrogated by preincubation of splenocytes for 24 hr with pertussis toxin (0.1-100 ng/mL). Pertussis toxin pretreatment of spleen cells was also found to directly abrogate cannabinoid inhibition of adenylate cyclase, as measured by forskolin-stimulated accumulation of intracellular cAMP. These results indicate that inhibition of the sRBC AFC response by cannabinoids is mediated, at least in part, by inhibition of adenylate cyclase through a pertussis toxin-sensitive Gi-protein coupled cannabinoid receptor. Additionally, these studies further support the premise that cAMP is an important mediator of lymphocyte activation.

Protective Effects of (–)-Epigallocatechin-3-Gallate on UVA- and UVB-Induced Skin Damage

It has been known that green tea and its components possess significant chemopreventive effects against chemical carcinogens and photo-caused skin tumor formation. In this study, the protective effects of (–)-epigallocatechin-3-gallate (EGCG), a major green tea catechin, on the ultraviolet (UV)-induced skin damage (photoaging) were studied in guinea pigs, hairless mice and human dermal fibroblast cultures. The lipid peroxidation was significantly reduced in the EGCG-treated group. The amount of lipid peroxides produced in the control and EGCG treated group were 838 ± 144 and 286 ± 57 nmol/mg at 18 h after UV irradiation, respectively. UVB-induced erythema was also significantly reduced in the EGCG treated group. The erythema relative index of the control and the EGCG treated group were 311 ± 45 and 191 ± 49 at 16 h after UV irradiation, respectively. EGCG treatment reduced UVA-induced skin damage (roughness and sagginess) and protected from the decrease of dermal collagen in hairless mouse skin. EGCG treatment blocked the UV-induced increase of collagen secretion and collagenase mRNA level in fibroblast culture. The nuclear transcription factors NF-ĸB and AP-1 binding activities were also inhibited by EGCG treatment.

Dexamethasone inhibits IL-1β gene expression in LPS-stimulated RAW 264.7 cells by blocking NF-κB/Rel and AP-1 activation

In the present study, the mechanism by which dexamethasone (DEX) inhibited IL-1beta gene expression in bacterial lipopolysaccharide (LPS)-activated RAW 264.7 cells was investigated. The decrease in LPS-induced IL-1beta mRNA expression was demonstrated by quantitative reverse transcription polymerase chain reaction (RT-PCR). Since the promoter in IL-1beta gene contains binding motifs for NF-kappaB/Rel, AP-1, NF-IL6, and CREB/ATF, which appear to be important in LPS-mediated IL-1beta induction, the effects of DEX on the activation of these transcription factors were examined. Treatment of DEX to RAW 264.7 cells induced a dose-related inhibition of NF-kappaB/Rel and AP-1 in chloramphenicol acetyltransferase activity, while neither NF-IL6 nor CREB/ATF activation was affected by DEX. Treatment of RAW 264.7 cells with DEX inhibited DNA binding of NF-kappaB/Rel and AP-1 proteins to their cognate DNA sites as measured by electrophoretic mobility shift assay (EMSA). DEX treatment caused a significant reduction in nuclear c-rel, p65, and p50 protein contents, and these decreases were paralleled by the accumulation of cytoplasmic c-rel, p65, and p50. DEX treatment of RAW 264.7 cells did not inhibit the nuclear translocation of c-jun and c-fos. We found that the inhibition of IL-1beta production by DEX is not related to p38, which is important in the IL-1beta induction. These results suggest that DEX may inhibit IL-1beta gene expression by a mechanism involving the blocking of LPS-induced NF-kappaB/Rel and AP-1 activation.

Suppression of IL-8 gene expression by radicicol is mediated through the inhibition of ERK1/2 and p38 signaling and negative regulation of NF-κB and AP-1

We show that radicicol, an anti-fungal agent, inhibits interleukin-8 (IL-8) production by the human monocyte line THP-1 in response to phorbol-12-myristate-13-acetate/lipopolysaccharide (PMA/LPS). IL-8 is a potent chemokine and needs for an optimal immune response--such as inflammation by activation of neutrophils. The decrease in PMA/LPS-induced IL-8 mRNA expression was demonstrated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Since the promoter in IL-8 gene contains binding motifs for NF-KB, AP-1. and NF-IL6, which appear to be important in IL-8 induction, the effects of radicicol on the activation of these transcription factors were examined. Treatment of radicicol to THP-1 cells produced a strong inhibition of NF-KB and AP-1, while NF-IL6 was not significantly affected by radicicol. Western blot analysis showed that radicicol inhibited the phosphorylation and phosphotransferase activities of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38. PD98059 and SB203580, known as a specific inhibitor of MEKI and p38 kinase, respectively, inhibited IL-8 gene expression showing that both of the kinase pathways are involved in IL-8 regulation in human monocytes. Collectively, this series of experiments indicates that radicicol inhibits IL-8 gene expression by blocking ERK1/2 and p38 signaling.

Suppression of the interleukin-2 gene expression by aflatoxin B1 is mediated through the down-regulation of the NF-AT and AP-1 transcription factors

The effect of aflatoxin B1 (AFB1) on the interleukin-2 (IL-2) gene expression was investigated in thymocytes of B6C3F1 mice, Jurkat E6-1 human T-cell leukemia, and EL4.IL-2 murine thymoma. AFB1 inhibited the phorbol-12myristate-13-acetate/i6nomycin (PMA/Io)-induced IL-2 mRNA expression in the murine thymocytes and Jurkat E6-1 cells as determined by qualitative RT-PCR, while no effect was observed in the EL4.IL-2 cells. Electrophoretic mobility shift assay indicated that AFB1 treatment showed an inhibition of the NF-AT and AP-1 DNA binding in PMA/Io-stimulated thymocytes and Jurkat E6-1 cells. No effect was observed on the Oct and NF-kappaB DNA binding. Employing a reporter gene expression system with p(NF-AT)3-CAT and p(AP-1)3-CAT, treatment with AFB1 to the transfected Jurkat E6-1 cells also showed an inhibition of the PMA/Io-induced NF-AT/CAT and AP-1/CAT activities. These results suggest that suppression of the IL-2 gene expression by AFB1 is mediated through the down-regulation of the NF-AT and AP-1 activation.

2-Amino-3-methylimidazo[4,5-f]quinoline inhibits nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells by blocking p38 kinase activation

We show that 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a heterocyclic amine, significantly inhibits nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages. The decrease in NO production was found to correlate well with a decrease in inducible NO synthase (iNOS) mRNA expression as demonstrated by Northern blot analysis. Treatment of RAW 264.7 cells with IQ selectively inhibited the activation of NF-kappaB/Rel, an important transcription factor of iNOS gene expression, while neither AP-1 nor Oct was affected by IQ. Since iNOS transcription has been shown recently to be under the control of the p38 kinase signaling cascade, we assessed the effect of IQ on p38 kinase activation. Treatment of RAW 264.7 with IQ inhibited LPS-stimulated p38 kinase phosphorylation in a dose-related manner. IQ also inhibited the p38 kinase activity. Collectively, this series of experiments indicates that IQ inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells. Based on our findings, the most likely mechanism that can account for this biological effect involves the negative regulation of NF-kappaB/Rel and p38 kinase pathway.

Production of DNA single-strand breaks in unstimulated splenocytes by dimethylnitrosamine

In an attempt to elucidate the mechanism whereby primary hepatocytes, but not liver S9 homogenates, generate immunosupprssive metabolites of dimethylnitrosamine (DMN), the production of DNA single-strand breaks (SSB) in unstimulated splenocytes was investigated with alkaline-elution analysis. Both hepatocytes and S9 homogenates induced SSB in cultured splenocytes by DMN - minimum detectable doses with the two metabolic activation systems (MAS) were 1 microM and 5 mM, respectively. DNA elution profiles were linear in splenocytes co-cultured with DMN and hepatocytes and convex in splenocytes incubated with DMN and S9 homogenates. Aminoacetonitrile (AAN; 10 mM), a DMN demethylase inhibitor, reversed SSB in splenocytes when incubated with either MAS. Addition of exogenous calf-thymus DNA to the hepatocyte co-culture medium did not affect the production of SSB. Rocking the hepatocyte-splenocyte cultures changed the elution profile from linear to convex. All of these treatments have been previously shown to block the immunosuppression by DMN in the hepatocyte co-culture system. These results indicate that the immunosuppression by DMN is not related to DNA damage, as measured by the production of SSB, and suggest that the metabolism of DMN to intermediates capable of producing genotoxicity and immunotoxicity may be qualitatively and/or quantitatively different.

The subunit composition of Portunus trituberculatus hemocyanin polymers

The subunit composition of isolated polymeric forms of Portunus trituberculatus hemocyanin were analysed by immunological techniques. The dodecamers contain four monomeric subunits corresponding to subunits I, II, III and IV, whereas the hexamers are devoid of subunit IV. These results suggest that subunit IV is required as a joining piece for the assembly of dodecamers.

Expression of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P4501A1 in human splenic lymphocyte cultures

The induction of cytochrome P4501A1 (P4501A1) and P4501A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in human splenic lymphocytes cultures. EROD activity was induced by TCDD in mitogen (phytohemagglutinin and pokeweed mitogen) stimulated blast cells but not in the resting cells. TCDD markedly induced EROD activity in a dose- and time-dependent manner. The expression of P4501A1 mRNA was increased by TCDD in mitogen-stimulated cells as detected by Northern blot analysis. These findings support the conclusion that TCDD induced the expression of P4501A1 gene, resulting in increased EROD activity in mitogen-stimulated human splenic lymphocytes cultures.

2-Acetylaminofluorene inhibits the activation of immune responses by blocking cell cycle progression at G1 phase.

Abstract2-Acetylaminofluorene (AAF) inhibited in a dose dependent manner mouse spleen cell blastogenesis in response to phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Io) activation, the T-cell lectin, concanavalin A (Con A), and following stimulation by alloantigens as measured by the mixed lymphocyte response (MRL). AAF also markedly suppressed the T-cell dependent antibody forming cell (AFC) response to sRBC. AAF was most inhibitory on both the sRBC IgM AFC response and Con A stimulated proliferation when added during the first 24 h following initiation of culture. Direct addition of high concentrations of AAF (100 μM) to spleen cell cultures at 48 h following Con A stimulation produced a very modes inhibition (<20%) of T-cell proliferation as compared to 90% when added at the time cultures were initiated. Similarly, AAF (75 and 100 μM) produced a greater than 80% inhibition of the in vitro AFC response when spleen cells were sensitized with antigen in presence of AAF. In contrast, no inhibition of the IgM AFC response was produced when AAF (75μM) was added to spleen cell cultures 48 or 72 h after antigen sensitization. Con A-triggered cell-cycle progression was attenuated at the G1 stage by the addition of AAF (50 and 100 μM) with no inhibition of S to G2/M phase transition. These results suggest that the mechanism of AAF-mediated immune suppression is through a blockade of cell cycle progression from G1 to S phase.