Search

(5R)-5-Hydroxytriptolide (LLDT-8) displays strong immunosuppressive activities both in vitro and in vivo in our previous studies. This study aims to investigate whether LLDT-8 has antiarthritic potential in a murine model of type II bovine collagen (CII)-induced arthritis (CIA) and to show the mechanism(s) of LLDT-8 action. DBA/1 mice were immunized with CII to induce arthritis and administered with LLDT-8. The severity of arthritis was evaluated according to the clinical score and joint damage. The effects of LLDT-8 on immune responses were determined by measurement of serum antibody levels, lymphocyte proliferation assay, cytokine assay, nitric oxide (NO) production, arginase activity assays, fluorescence-activated cell sorting analysis of splenic Mac-1(+) cells, as well as polymerase chain reaction analysis for interferon-gamma (IFN-gamma)-related gene expression. We showed that LLDT-8 treatment significantly reduced the incidence and severity of CIA. The preventive and therapeutic effects of LLDT-8 are associated with 1) reduction of serum anti-CII immunoglobulin (Ig) G, IgG2a, and IgG1 levels; 2) inhibition of CII-specific lymphocyte proliferation, IFN-gamma and interleukin-2 production; 3) blockade of gene expressions in IFN-gamma signaling, including IFN-gamma production pathways [signal transducer and activator of transcription (STAT) 1, T-box transcription factor, interleukin 12Rbeta2, and STAT4] and IFN-gamma-induced chemokine transcription [macrophage inflammatory protein (Mip)-1alpha, Mip-1beta, regulated on activation normally T cell expressed and secreted, and inducible protein 10]; and 4) retardation of the abnormal increase of NO via IFN-gamma/STAT1/interferon regulatory factor 1/inducible nitric-oxide synthase pathway and arginase activity. Moreover, the mRNA transcription of chemokine receptors was also suppressed [including C-C chemokine receptor (CCR) 1, CCR5, and C-X-C chemokine receptor 3]. In conclusion, our data suggest that the antiarthritic effect of LLDT-8 is closely related to the blockade of IFN-gamma signaling. LLDT-8 may have a therapeutic value in the treatment of rheumatoid arthritis.

The reversible S-adenosyl-l-homocysteine (AdoHcy) hydrolase inhibitor methyl 4-(adenin-9-yl)-2-hydroxybutanoate (DZ2002) suppresses macrophage activation and function. The effects of DZ2002 on T cell function, however, are still unclear. Here, we examined whether DZ2002 alters type 1 helper T cell (Th1) and/or type 2 helper T cell (Th2) immune responses, and whether these effects are associated with both the inhibition of AdoHcy hydrolase and intracellular elevation of endogenous AdoHcy. Male C57BL/6 mice immunized with ovalbumin (OVA) were treated with DZ2002 (1, 5, and 25 mg/kg/day) after which lymphocyte proliferation, cytokine production, and IgG responses to OVA were monitored. Administration of DZ2002 dose dependently suppressed OVA-specific lymphocyte proliferation and anti-OVA IgG production compared with controls. Interleukin (IL)-2 and interferon (IFN)-gamma as well as anti-OVA IgG2a and IgG3, indicators of Th1 immune responses, were markedly decreased in mice treated with DZ2002, whereas IL-4 and anti-OVA IgG1, indicators of Th2 immune responses, were only mildly suppressed. AdoHcy hydrolase activity in spleens of DZ2002-treated mice was substantially blocked, and not surprisingly, AdoHcy levels were significantly elevated compared with controls. Finally, similar immunosuppressive effects were also observed in mice treated with AdoHcy. These data strongly indicate that DZ2002 suppresses antigen-induced specific immune responses, particularly Th1 responses, through inhibition of AdoHcy hydrolase and elevation of endogenous AdoHcy.

Periploca sepium Bge, a traditional Chinese herb medicine, is used for treating rheumatoid arthritis in China. Followed the bioactivity-guided isolation, the most potent immunosuppressive compound, periplocoside E (PSE), a pregnane glycoside, had been identified from P. sepium Bge. We investigated the immunosuppressive effects of PSE in vitro and in vivo. The results showed that PSE in a dose-dependent manner significantly inhibited the proliferation of splenocytes induced by concanavalin A and mixed lymphocyte culture reaction at no cytotoxic concentrations (<5 muM). Administration of PSE suppressed a delayed-type hypersensitivity reaction, and ovalbumin (OVA) induced antigen-specific immune responses in mice. In vivo treatment with PSE dose dependently suppressed OVA-induced proliferation and cytokine [interleukin (IL)-2 and interferon (IFN)-gamma] production from splenocytes in vitro. Purified T cells from OVA-immunized mice with PSE treatment showed its low ability for activation by OVA plus normal antigen presenting cell stimulation again in vitro. Further studies showed PSE dose dependently inhibited anti-CD3-induced primary T cell proliferation, activation for IL-2Ralpha (CD25) expression, and cytokine (IFN-gamma and IL-2) production also at the transcriptional level. PSE was highly specific and significantly inhibited the activation of extracellular signal-regulated kinase and Jun N-terminal kinase, whereas activation of p38 was not affected in T cells stimulated with anti-CD3. These results demonstrated that PSE is an immunosuppressive compound in P. sepium Bge, which directly inhibits T cell activation in vitro and in vivo. This study provided evidence to understand the therapeutic effects of P. sepium Bge and indicated that this herb is appropriate for treatment of T cell-mediated disorders, such as autoimmune diseases.

(5R)-5-Hydroxytriptolide (LLDT-8) is a novel analog of triptolide that has antiarthritic, hepatoprotective, and antiallogenic transplantation-rejective effects. In the present study, we report that LLDT-8 inhibited nitric oxide (NO) production and inducible nitric-oxide synthase (iNOS) expression in macrophages. LLDT-8 significantly attenuated NO production, in a dose-dependent manner, in primary peritoneal macrophages and a macrophage cell line of Raw 264.7 cells following stimulation with interferon (IFN)-gamma, lipopolysaccharide (LPS), and IFN-gamma plus LPS. It also reduced the production of tumor necrosis factor-alpha from LPS-stimulated Raw 264.7 cells. To further elucidate the mechanism responsible for the inhibition of NO, we examined the effect of LLDT-8 on IFN-gamma and LPS-induced iNOS expression. Indeed, LLDT-8 prevented NO generation by inhibiting iNOS expression at mRNA level and protein level, rather than by interfering its enzymatic activity. In IFN-gamma-stimulated Raw 264.7 cells, LLDT-8 suppressed the gene transcription of signal transducer and activator of transcription 1alpha and interferon regulatory factor (IRF)-1, but it displayed no apparent effect on IFN-gamma receptor level on cell surface. After LPS challenge, LLDT-8 further abrogated the expression of LPS receptor complex, including CD14, Toll-like receptor 4, and myeloid differentiation protein-2; decreased the LPS-induced phosphorylation of stress-activated protein kinase/c-Jun NH(2)-terminal kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase (MAPK); retarded the degradation of IkappaBalpha; and ameliorated the DNA binding activity of nuclear factor-kappaB (NF-kappaB) to nuclear proteins that accounts for transcriptional regulation of iNOS. Taken together, these results suggest that LLDT-8 reduces NO production and iNOS expression by inhibiting IFN-gamma-triggered IRF-1 expression and LPS-triggered MAPK phosphorylation and NF-kappaB activation.

Lymphocytes depend on transmethylation reactions for efficient activation and function. These reactions are primarily catalyzed by S-adenosylmethionine-dependent methyltransferases, which convert S-adenosylmethionine to S-adenosyl-L-homocysteine. S-adenosyl-L-homocysteine is then hydrolyzed by S-adenosyl-L-homocysteine hydrolase to prevent feedback inhibition of transmethylation reactions. By impeding S-adenosyl-L-homocysteine hydrolase, a build-up of S-adenosyl-L-homocysteine occurs, and most intracellular transmethylation reactions cease. Thus, a nontoxic inhibitor of this enzyme might be a useful immunosuppressive therapeutic agent. We identified a potent reversible type III inhibitor of S-adenosyl-L-homocysteine hydrolase, DZ2002 [methyl 4-(adenin-9-yl)-2-hydroxybutanoate], and determined its cytotoxic and immunologic effects. We demonstrated that DZ2002 blocked S-adenosyl-L-homocysteine hydrolase more effectively than a type I inhibitor, but cytotoxicity from DZ2002 was greatly reduced. Although DZ2002 did not prevent concanavalin A-induced T cell proliferation or interleukin (IL)-2 production, it significantly reduced both a mixed lymphocyte reaction and IL-12 production from in vitro-stimulated splenocytes. In addition, levels of CD80 and CD86 on human monocytic THP-1 cells were decreased in a dose-dependent manner in the presence of 0.1 to 10 microM DZ2002, and decreases were also seen in IL-12 and tumor necrosis factor-alpha production from both mouse thioglycollate-stimulated peritoneal macrophages and THP-1 cells. In vivo, DZ2002 significantly suppressed a delayed-type hypersensitivity reaction as well as antibody secretion. We conclude that DZ2002's immunosuppressive effects are likely not solely attributed to T cell inhibition but also to the obstruction of macrophage activation and function through reductions in cytokine output and/or T cell costimulation. These data suggest an important dual role for the S-adenosyl-l-homocysteine hydrolase in both macrophage and T cell function.