Katsutoshi Komuro

IRF-8/ICSBP and IRF-1 cooperatively stimulate mouse IL-12 promoter activity in macrophages

IRF‐8/ICSBP and IRF‐1 are IRF family members whose expression is induced in response to IFN‐γ in macrophages. IL‐12 is a cytokine produced in macrophages that plays a critical role in host defense. IFN‐γ and bacterial lipopolysaccharide (LPS) induce IL‐12p40 transcription, which is necessary for the production of IL‐12. We have previously shown that IL‐12p40 expression is impaired in ICSBP‐deficient mice and that transfection of ICSBP together with IRF‐1 can activate IL‐12p40 expression in mouse macrophage cells. To further study the role of ICSBP and IRF‐1, we investigated murine IL‐12p40 promoter activity in the macrophage cell line RAW 264.7. We show here that co‐transfection of ICSBP and IRF‐1 synergistically stimulates IL‐12 promoter activity to a level comparable to that induced by IFN‐γ/LPS. Mutation of the Ets or NFκB site previously shown to be important for IL‐12p40 transcription did not abolish the activation by ICSBP and IRF‐1. However, mutation of the ISRE‐like site found downstream from the NFκB and C/EBP sites abrogated the activation by ICSBP and IRF‐1. Together, these results indicate that ICSBP and IRF‐1 cooperatively stimulate murine IL‐12 transcription through a novel regulatory element in the murine promoter.

Nucleolin is involved in interferon regulatory factor-2-dependent transcriptional activation

We have previously shown that interferon regulatory factor-2 (IRF-2) is acetylated in a cell growth-dependent manner, which enables it to contribute to the transcription of cell growth-regulated promoters. To clarify the function of acetylation of IRF-2, we investigated the proteins that associate with acetylated IRF-2. In 293T cells, the transfection of p300/CBP-associated factor (PCAF) enhanced the acetylation of IRF-2. In cells transfected with both IRF-2 and PCAF, IRF-2 associated with endogenous nucleolin, while in contrast, minimal association was observed when IRF-2 was transfected with a PCAF histone acetyl transferase (HAT) deletion mutant. In a pull-down experiment using stable transfectants, acetylation-defective mutant IRF-2 (IRF-2K75R) recruited nucleolin to a much lesser extent than wild-type IRF-2, suggesting that nucleolin preferentially associates with acetylated IRF-2. Nucleolin in the presence of PCAF enhanced IRF-2-dependent H4 promoter activity in NIH3T3 cells. Nucleolin knock-down using siRNA reduced the IRF-2/PCAF-mediated promoter activity. Chromatin immunoprecipitation analysis indicated that PCAF transfection increased nucleolin binding to IRF-2 bound to the H4 promoter. We conclude that nucleolin is recruited to acetylated IRF-2, thereby contributing to gene regulation crucial for the control of cell growth.

Ovalbumin-Liposome Conjugate Induces IgG but Not IgE Antibody Production

Antibody response after immunization with surface-coupled ovalbumin (OVA) of liposomes was investigated in mice. OVA was coupled to the surface of liposome via amino groups using glutaraldehyde. OVA-liposome conjugate induced a significant anti-OVA IgG antibody production in mice. However, no IgE antibody production specific for OVA was observed. Immunization with OVA-liposome induced IgE-specific unresponsiveness even after the subsequent challenge with OVA adsorbed with with aluminium hydroxide (OVA-alum), which induces a high level of IgE antibody production. Furthermore, following the primary immunization with OVA-alum, a secondary challenge with OVA-liposome boosted anti-OVA IgG but not anti-OVA IgE antibody production. These results show the potential of the antigen-liposome conjugate for the development of a vaccine with the least allergic reaction and also for the application of immunotherapy.

T Cell-Independent Regulation of IgE Antibody Production Induced by Surface-Linked Liposomal Antigen

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.

Antigen–Specific, IgE–Selective Unresponsiveness Induced by Antigen–Liposome Conjugates

Background: We have previously reported that ovalbumin (OVA) coupled with liposome via glutaraldehyde (GA) induced OVA–specific– and IgE–selective unresponsiveness in mice. Methods: In this study, OVA–liposome conjugates were made using four different coupling protocols: via GA, N–(6–maleimidocaproyloxy) succinimide (EMCS), disuccinimidyl suberate (DSS) and N–succimidyl–3(2–pyridyldithio)propionate (SPDP) and the induction of antigen–specific IgG and IgE antibody production was investigated for each. In addition, antigen–specific cytokine production by spleen cells of mice immunized either with OVA–liposome or with OVA adsorbed with aluminum hydroxide was investigated. Results: OVA–liposome conjugates coupled via GA or DSS did not induce anti–OVA IgE antibody production but induced substantial anti–OVA IgG antibody production. On the other hand, the induction of anti–OVA IgE unresponsiveness by OVA–liposome conjugates coupled via EMCS or SPDP was incomplete. The amount of interleukin 4 (IL–4) produced by spleen cells stimulated in vitro with OVA correlated well with anti–OVA IgE antibody production in donor mice. However, the production of no other cytokine, i.e., IL–2, IL–5, IL–10 or interferon–γ, was correlated with in vivo IgE antibody production. Conclusion: OVA–liposome coupled via GA or DSS induced complete suppression of anti–OVA IgE production. The results in this study further suggest that the regulation of IgE antibody production does not neccessarily correlate with so–called Th1 cytokine production.

Protection of Monkeys against Shiga Toxin Induced by Shiga Toxin-Liposome Conjugates

Background: We previously reported that the purified Shiga toxins (Stx) Stx1 and Stx2, when coupled with liposomes, induced substantial production of anti-Stx1 and anti-Stx2 IgG antibody, respectively, in mice. The levels of anti-Stx antibody in the sera of mice immune to Stx-liposome correlated well with the protection against subsequent challenge with Stx. Furthermore, mice immunized with a mixture of Stx1-liposome and Stx2-liposome were successfully protected against oral infection with cytotoxin-producing Escherichia coli O157:H7. Methods: In this study, the induction of protection against Stx2 by Stx2-liposomes was evaluated in monkeys. Results: Stx2-liposomes induced a substantial amount of anti-Stx2 IgG antibodies as well as Stx2 neutralizing antibodies in monkeys. Test monkeys were successfully protected against challenge with lethal doses of Stx2. Moreover, these monkeys showed no apparent symptoms, while nonimmunized control monkeys died within 4 days with hemorrhagic gastroenteritis and renal disorder. In addition, as shown by other cases involving antigen-liposome conjugates, Stx2-liposome did not induce anti-Stx2 IgE antibody production, though it stimulated the production of a substantial amount of anti-Stx2 IgG antibodies. Conclusion: These results suggest that Stx-liposome conjugates may serve as candidate vaccines to induce protection against death caused by cytotoxin-producing E. coli infection.

Induction of Protection against Oral Infection with Cytotoxin–Producing Escherichia coli O157:H7 in Mice by Shiga–like Toxin–Liposome Conjugate

We have previously reported that purified Shiga–like toxins (SLT), SLT–I and SLT–II coupled with liposomes induced a substantial amount of anti–SLT–I and anti–SLT–II IgG antibody production, respectively, in mice. The levels of anti–SLT antibody in the sera of SLT–liposome–immune mice correlated well with the protection against subsequent challenge with SLT. In this study, mice were immunized intraperitoneally with the mixture of SLT–I–liposome and SLT–II–liposome and protection against oral infection with cytotoxin–producing Escherichia coli O157:H7 was evaluated. All of the mice that received immunization with the mixture of SLT–I–liposome and SLT–II–liposome were protected against subsequent intravenous challenge with 10 LD50 of either SLT–I or SLT–II. Eight weeks after primary immunization, mice were inoculated intragastrically with 109 CFU of E. coli O157:H7 strain 96–60. All SLT–liposome–immune mice tested survived without any apparent symptom while control mice died within 5 days. In addition, as shown by other antigen–liposome conjugates, SLT–liposome induced undetectable anti–SLT IgE antibody production while they induced substantial amounts of anti–SLT IgG antibodies. These results suggest that SLT–liposome conjugate may serve as a candidate vaccine that induces protection against cytotoxin–producing E. coli infection.

A highly efficient method for the site-specific integration of transfected plasmids into the genome of mammalian cells using purified retroviral integrase

Using purified bovine leukemia virus (BLV) integrase with liposome, we developed a highly efficient method for the site-specific integration of plasmid vectors into the genome of cultured mammalian cells. The presence of the BLV integrase recognition sequence (IRS) in both the host genome and the plasmid vector to be transfected was required for this integration. The integration occurred within the IRS pre-introduced into the host genome and resulted in a complete or partial deletion of the sequence and an adjacent drug-resistant gene. This site-specific integration was not observed upon transfection without the integrase or with vectors harboring no IRS. This novel method may be useful for manipulating a mammalian genome or for targeting a retroviral genome integrated into a virus-infected cell by using the virus-specific integrase and LTR sequence.

Macrophage Inhibition of Lymphocyte and Tumor Cell Growth Is Mediated by 25–Hydroxycholesterol in the Cell Membrane

We have previously reported that a lipid molecule in the membrane fraction of cloned macrophage hybridomas inhibited the growth of lymphocytes and several tumor cell lines. In this study, the inhibitory lipid molecule in the membrane fraction of macrophages was analyzed by thin–layer chromatography and identified as 25–hydroxycholesterol, a family of oxysterols. This conclusion was confirmed by analysis using gas chromatography–mass spectrometry. In addition, both 25–hydroxycholesterol and the lipid molecule recovered from macrophage cell membrane induced apoptosis of the murine T cell lymphoma, BW–5147. These results suggest that an oxysterol expressed in the macrophage cell membrane may participate in the regulation of cell growth through cell contact.

Induction of Protection against Tetanus Toxin in Mice by Tetanus Toxoid–Liposome Conjugate

Tetanus toxoid (Ttd) was coupled to liposomes via glutaraldehyde. Intraperitoneal injection in BALB/c mice with Ttd–liposomes induced a substantial amount of anti–Ttd IgG antibody production and an extremely low level of anti–Ttd IgE antibody production. Mice immunized with Ttd–liposomes were successfully protected against a subsequent challenge with a lethal dose of tetanus toxin (Ttx). On the other hand, aluminum hydroxide–adsorbed Ttd (Ttd–alum) and plain Ttd solution induced the production of both IgG and IgE antibodies against Ttd. Moreover, secondary immunization with Ttd–liposomes in mice, in which anti–Ttd IgE antibody production was induced by Ttd–alum led to enhanced anti–Ttd IgG and a limited anti–Ttd IgE antibody production. When Ttd–liposome preparation was lyophilized, the efficacy of Ttd–liposomes was maintained for 6 months at 37°C, suggesting that this vaccine preparation would be stable without refrigeration. These results demonstrate the potential ability of Ttd–liposome conjugates to produce a tetanus vaccine which provides protection against (Ttx) while inducing the least amount of anti–Ttd IgE antibodies.