Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha} induction.

Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-alpha in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7 signaling pathway. IRAK-1 directly bound and phosphorylated IRF7 in vitro. The kinase activity of IRAK-1 was necessary for transcriptional activation of IRF7. TLR7- and TLR9-mediated IFN-alpha production was abolished in Irak-1-deficient mice, whereas inflammatory cytokine production was not impaired. Despite normal activation of NF-kappaB and mitogen-activated protein kinases, IRF7 was not activated by a TLR9 ligand in Irak-1-deficient pDCs. These results indicated that IRAK-1 is a specific regulator for TLR7- and TLR9-mediated IFN-alpha induction in pDCs.

The nuclear IkappaB protein IkappaBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria.

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMphi). CLPMphi of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMphi gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMphi. These genes included nuclear IkappaB proteins such as Bcl-3 and IkappaBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-alpha production, we analyzed the role of IkappaBNS in macrophages. Lentiviral introduction of IkappaBNS resulted in impaired LPS-induced IL-6 production, but not TNF-alpha production in the murine macrophage cell line RAW264.7. IkappaBNS expression led to constitutive and intense DNA binding of NF-kappaB p50/p50 homodimers. IkappaBNS was recruited to the IL-6 promoter, but not to the TNF-alpha promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IkappaBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-alpha. Thus, IkappaBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IkappaB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.

Regulation of lipopolysaccharide-inducible genes by MyD88 and Toll/IL-1 domain containing adaptor inducing IFN-beta.

Macrophages recognize lipopolysaccharide (LPS) by Toll-like receptor 4 and activate inflammatory responses by inducing expression of various genes. TLR4 activates intracellular signaling pathways via TIR domain containing adaptor molecules, MyD88, and Toll/IL-1 domain containing adaptor inducing IFN-beta (TRIF). Although macrophages lacking MyD88 or TRIF showed impaired cytokine production, activation of intracellular signaling molecules still occurred in response to LPS in these cells. In the present study, we implemented cDNA microarrays to investigate the contribution of MyD88 and TRIF in gene expression induced by LPS stimulation. Whereas wild-type macrophages induced 148 genes in response to LPS, macrophages lacking both MyD88 and TRIF did not upregulate any genes in response to LPS. Surprisingly, 80 LPS-inducible genes were redundantly regulated by either MyD88 or TRIF. In contrast, proinflammatory cytokines and chemokines were critically regulated by MyD88 or TRIF alone. Genes critically regulated by MyD88 alone tend to be induced quickly after LPS stimulation and regulated by mRNA stability as well as transcription. Genes known to be induced by type I interferons were simply dependent on TRIF for their expression. Taken together, MyD88 and TRIF play both redundant and distinct roles in LPS-induced gene expression.

Circulating auto-antibody against hepatoma-derived growth factor (HDGF) in patients with ulcerative colitis.

BACKGROUND/AIMS: Various circulating auto-antibodies have been reported in patients with ulcerative colitis. Hepatoma-derived growth factor (HDGF) is a mitogen, localized dominantly in the nucleus of proliferating cells. In this study, we demonstrated the circulating anti-HDGF auto-antibody and investigated its clinical roles in patients with ulcerative colitis. METHODOLOGY: Anti-HDGF IgG antibodies were measured by the enzyme-linked immunosorbent assay with recombinant HDGF in 20 healthy volunteers and 40 patients with ulcerative colitis. RESULTS: Circulating anti-HDGF antibody was detected in the serum of a patient with total colitis by Western blotting. Anti-HDGF auto-antibodies were detected at 65.6% in the serum of patients with total/left-sided colitis, compared with healthy subjects at 10%. During active stage, the circulating anti-HDGF auto-antibodies were detected at a higher frequency of 78.3% than those in remission stage at 37.5%. Furthermore, the titers during active colitis were higher than those during the remission stage. Anti-HDGF auto-antibodies were not detected in any patients with proctitis. CONCLUSIONS: These findings suggest that anti-HDGF auto-antibodies in the serum of patients with ulcerative colitis would help to classify the total/left-sided colitis from proctitis, and the serial measurement of the titer would also be a good marker for the active colitis.

A pilot study of combination therapy with initial high-dose interferon and amantadine hydrochloride for patients with chronic hepatitis C with the genotype 1b virus.

BACKGROUND/AIMS: Interferon monotherapy for patients with chronic hepatitis C has been suboptimal. We studied the effect of the combination therapy of an initial high-dose of interferon and amantadine. METHODOLOGY: We investigated the virological response of 20 patients with naive chronic hepatitis C with a high viral load of the genotype 1b virus. Seven patients were administered 6MU of interferon-beta once daily for 6 weeks and then thrice weekly for 20 weeks, and 13 were administered 6 MU of interferon-beta daily for 4 or 6 weeks and then 10 MU of natural interferon-alpha thrice weekly for 22 or 20 weeks. All patients were treated with amantadine hydrochloride (100 mg/day) for 26 weeks during interferon administration. RESULTS: The complete response, transient response and no response rate were 15.0%, 60.0%, and 25%, respectively. After daily administration of interferon-beta intravenously, 19 patients (95.0%) showed negative tests for serum HCV-RNA by the polymerase chain reaction method. At the end of treatment, the serum HCV-RNA was not detected in any patients treated with daily interferon-beta and intermittent interferon-alpha with amantadine. At 6-month follow-up, three patients had eradicated HCV-RNA, who were in the group of daily interferon-beta and intermittent interferon-alpha with amantadine. In the patients treated with daily interferon-beta and intermittent interferon-alpha with amantadine, the complete response, transient response and no response rates were 23.1%,-76.9% and 0%, respectively. CONCLUSIONS: These findings suggest that the combination of an initial high-dose interferon and amantadine shows promising effects on the eradication of HCV-RNA in the chronic hepatitis C patients with a high viral load of the genotype 1b virus.

Hepatoma-derived growth factor induces tumorigenesis in vivo through both direct angiogenic activity and induction of vascular endothelial growth factor.

Hepatoma-derived growth factor (HDGF) is highly expressed in tumor cells, and stimulates their proliferation. In the present study, we investigated the role of HDGF in tumorigenesis and elucidated the mechanism of action. Stable transfectants of NIH3T3 cells overexpressing HDGF did not show significant anchorage-independent growth in soft agar assay. However, these stable transfectants overexpressing HDGF generated sarcomatous tumors in nude mice. These tumors were red-colored macroscopically, and histologically showed a rich vascularity. Immunohistochemical analysis using CD31 antibody showed new vessel formation. Recombinant HDGF stimulated proliferation of human umbilical vein endothelial cells in a dose-dependent manner, and stimulated tubule formation. Furthermore, vascular endothelial growth factor (VEGF) was detected immunohistochemically in the tumor tissues. Transient expression of HDGF induced both VEGF gene and protein expression as demonstrated by a reporter assay using VEGF gene promoter. The administration of anti-VEGF neutralizing antibody significantly suppressed, but did not block, the tumor growth of HDGF-overexpressing cells in nude mice. Thus, these findings suggested that HDGF-induced tumor formation in vivo involves induction of VEGF as well as direct angiogenic activity.

The combination therapy of interferon and amantadine hydrochloride for patients with chronic hepatitis C.

BACKGROUND/AIMS: The effect of interferon treatment for chronic hepatitis C patients with genotype 1b virus has been suboptimal. We studied the effect of the combination therapy of interferon and amantadine on patients with a high serum viral load of genotype 1b virus. METHODOLOGY: We studied the virological response of naive chronic hepatitis C patients with a high viral load of genotype 1b virus (4.5 log copies/50 microL or 100 kcopies/mL and higher) during interferon and amantadine administration for 6 months and 6 months after the end of treatment. Twenty patients were treated with interferon alone (natural interferon-beta 6 MU daily for 6 weeks and thrice-a-week for 20 weeks) for 26 weeks. Eleven patients were treated with the combination therapy of interferon and amantadine hydrochloride (100 mg orally daily) for 26 weeks. RESULTS: After daily administration of interferon-beta intravenously once a day for 6 weeks, all patients showed the negative tests of serum HCV-RNA by polymerase-chain-reaction methods by the combination therapy, while 13 patients (65.0%) showed the negative tests by interferon alone (p = 0.0257). At the end of treatment, serum HCV-RNA were not detected in 54.5% of patients treated with interferon and amantadine, while it was detected in 50.0% of patients treated with interferon alone. At 6 months follow-up, only one patient (9.1%) could eradicate HCV-RNA in patients with interferon and amantadine, while no patient could with interferon monotherapy (not significantly). CONCLUSIONS: Amantadine hydrochloride has the additive effects to interferon treatment on the virological responses of serum HCV-RNA during a co-administration, although the combination therapy has not shown a significantly promising effect on the eradication of HCV-RNA in the patients with chronic hepatitis C with a high viral load of genotype 1b virus.

Anti-IL-6 receptor monoclonal antibody inhibits leukocyte recruitment and promotes T-cell apoptosis in a murine model of Crohn's disease.

BACKGROUND: To assess the contribution of IL-6 signaling to the physiopathology of Crohn's disease, we introduced anti-IL-6 receptor monoclonal antibody to a murine colitis model. METHODS: Colitis was induced in C.B-17-scid mice to which were transferred CD45RBhigh CD4+ T cells from Balb/c mice. Anti-IL-6 receptor monoclonal antibody or rat IgG was given intraperitoneally after T-cell transfer, followed by weekly injection. Vascular adhesion molecules and inducible nitric oxide synthase were visualized by immunostaining. Cytokine expression was determined by RT-PCR, and apoptotic cells were determined by the TUNEL method. RESULTS: Mice treated with anti-IL-6 receptor monoclonal antibody showed normal growth while controls lost weight. Colitis was improved histologically with reduced infiltration of LFA-1+ monocytes/macrophages and VLA-4+ T cells. ICAM-1 and VCAM-1 expression in the colonic vascular endothelium was markedly suppressed by the treatment, whereas no significant difference was seen in MAdCAM-1. IFN-gamma, TNF-alpha, and IL-1beta mRNAs were markedly reduced, but no difference was observed in the expression of IL-4, IL-10, and TGF-beta. Inducible nitric oxide synthase was upregulated in the mucosa of colitic mice and downregulated in the treated mice. Apoptotic cells were very sparse despite massive CD4+ T-cell infiltration in colitic mice, whereas increased apoptosis was seen in the treated mice with an apparently reduced number of T cells. CONCLUSIONS: Anti-IL-6 receptor monoclonal antibody abrogated murine colitis. It effectively blocked the expression of adhesion molecules, thereby blocking leukocyte recruitment, and increased T-cell apoptosis. These results strongly suggest the therapeutic potential of anti-IL-6 receptor monoclonal antibody for human Crohn's disease.