Misako Asano

Functional evaluation of tumor‐infiltrating mononuclear cells. Detection of endogenous interferon‐γ and tumor necrosis factor‐α in human colorectal adenocarcinomas

Quantitative evaluation of the levels of interferon‐γ (IFN‐γ) and tumor necrosis factor‐α (TNF‐α) in the extracts of tumors and their corresponding normal tissues resected from 43 patients with colorectal adenocarcinoma was done using solid‐phase, sandwich radioimmunoassay. The levels of both IFN‐γ and TNF‐α detected in the tumor tissues were higher than those in the corresponding normal colorectal tissues obtained from each patient. A significant negative correlation was observed between the level of IFN‐γ and TNF‐α in each tumor extract. The decrease of the level of IFN‐γ in the tumor correlated with the advance of clinical stage, and the levels of IFN‐γ of the patients with distant metastases were significantly lower than those of the patients without distant metastases. However, an increase in the level of TNF‐α correlated not only with an enlarged diameter but also with the extent of the primary tumor. Immunohistochemical staining of IFN‐γ and TNF‐α producing cells in tumor tissues showed that IFN‐γ was mainly produced by CD4 + CD8– T‐lymphocytes and TNF‐α was mainly produced by CD11c+ cells with macrophage‐like morphology. These results suggest that CD4+ T‐lymphocytes that produce IFN‐γ might play an important role in the antitumor response against cancer progression in human colorectal adenocarcinomas.

Detection of high levels of immunoreactive human beta-1 interferon in sera from HIV-infected patients

We have examined human interferon beta-1 (HuIFN-beta 1) levels in the sera from patients with various diseases by using a newly developed highly sensitive enzyme-linked immunosorbent assay system (ELISA). High levels of HuIFN-beta 1 were detected in the sera from Human Immunodeficiency Virus-I (HIV-I) infected patients, especially of asymptomatic carriers. The serum level of HuIFN-beta 1 may be a good indicator of HIV-I infection and of other immunological disorders.

Effects of 15-deoxyspergualin in vitro and in vivo on cytokine gene expression.

Reverse transcriptase-polymerase chain reaction showed that interleukin 3, IL-4, IL-5, IL-6, interferon-gamma and stem cell factor mRNA expression were higher in 15-deoxyspergualin-treated spleen cells than in control spleen cells. Increased IL-2 and IFN-gamma mRNA expression were observed in 15-deoxyspergualin-treated bone marrow cells. On the other hand, increased platelet counts in BALB/c-->C3H/He bone marrow chimeras were observed from days 20 to 33 in our previous work, when they were treated with 15-deoxyspergualin from days 14 to 25. In contrast, marked leukocytopenia and anemia were simultaneously observed, although a marked leukocytosis and a rapid recovery of anemia were observed on day 33 and thereafter. To analyze effects of 15-deoxyspergualin on hematopoiesis and the immune system, we examined mRNA expression in bone marrow and spleen cells from BALB/c-->C3H/He bone marrow chimeras treated with 15-deoxyspergualin from days 14 to 25. Reverse transcriptase-polymerase chain reaction showed that IL-3, IL-4, IL-6, stem cell factor, granulocyte colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor mRNA expression were higher in 15-deoxyspergualin-treated chimeras than in control chimeras, indicating that these cytokines are responsible for an enhancement of hematopoiesis. It was conceivable that IL-6 supported thrombopoiesis in concert with other cytokines. On the contrary, increased IFN-gamma, IL-2, IL-3, IL-4, and IL-10 mRNA expression may play an immunosuppressive role in vivo.

Sequential involvement of NK cells and CD8+ T cells in granuloma formation of Rhodococcus aurantiacus-infected mice

We investigated the effect of in vivo administration of antibodies against T‐cell subsets and natural killer (NK) cells on endogenous gamma interferon (IFN‐γ) production and granuloma formation in Rhodococcus aurantiacus‐infected mice. High titers of endogenous IFN‐γ were detected in the extracts of the livers and spleens during 24 hr of the infection, reaching the peak at 8 hr, and the IFN‐γ production was reduced by in vivo administration of anti‐NK 1.1 monoclonal antibody (MAb) or antibody against asialo GM1+ cells. Endogenous IFN‐γ declined until 2 days of the infection, then reappeared from 1 week and peaked at 3 weeks. Endogenous IFN‐γ at 1 and 3 weeks was reduced by in vivo administration of anti‐CD8 MAb, but not by anti‐CD4 MAb or anti‐NK 1.1 MAb. Granulomatous lesions in the livers and spleens began to appear from 1 week of the infection and developed in 3 weeks. In vivo administration of rat anti‐IFN‐γ MAb reduced the development of granulomas. In addition, granuloma formation was reduced by depletion of NK cells prior to the infection or depletion of CD8+ T cells at 1 week of the infection. Based on these findings, it is presumed that the biphasic production of IFN‐γ is attributable to NK cells in the early phase of the infection and CD8+ T cells in the phase of granuloma formation, and that granuloma formation is regulated by NK cells and CD8+ T cells through the secretion of endogenous IFN‐γ.

Reciprocal action of interferon-gamma and interleukin-4 promotes granulomatous inflammation induced by Rhodococcus aurantiacus in mice.

An intravenous injection of Rhodococcus aurantiacus to mice causes granulomatous inflammation dependent on endogenous interferon‐γ (IFN‐γ). The present study examined the role of endogenous interleukin‐4 (IL‐4) on granulomatous inflammation. Endogenous IL‐4 in the spleen extracts was not detected during the phase of granuloma formation by enzyme‐linked immunosorbent assay (ELISA). However, IL‐4 protein level was elevated during the phase of granuloma regression. IL‐4 mRNA expression in the livers and spleens was also elevated during the phase of granuloma regression. In addition, IL‐4 levels during the phase of granuloma formation were increased by treatment with anti‐IFN‐γ monoclonal antibody (mAb), suggesting that endogenous IFN‐γ might inhibit IL‐4 production during the phase of granuloma formation. Administration of anti‐IL‐4 mAb on weeks 3 and 4 after the inoculation inhibited the regression of granulomas and augumented IFN‐γ level at 5 weeks. Endogenous IFN‐γ was produced by CD4+ T cells during the phase of granuloma regression and endogenous IL‐4 was produced by both CD4+ and CD8+ T cells. These findings suggest that during the phase of granuloma formation endogenous IL‐4 might be inhibited by IFN‐γ, while during the phase of granuloma regression endogenous IL‐4 might play a crucial role in the reduction of granulomas and IFN‐γ production.

Theiler's virus is eliminated by a gamma-interferon-independent mechanism in the brain

Abstract The intravenous infection of Theilers virus GD VII strain causes acute encephalomyelitis in infected mice. To determine the cellular mechanism of resistance and interferon (IFN)-γ-producing cell populations, mononuclear cells isolated from tissues of the brain were analyzed by the flow cytometry method. Antibodies specific for CD3, CD4, CD8, T cell receptor (TCR)-αβ, and Asialo GM1 were used to deplete the corresponding cell populations in Theilers virus-infected mice. CD4+ lymphocytes and CD8+ lymphocytes infiltrated in the brains of infected mice from 5 days postinfection (p.i.). The number of CD3+/TCR-γδ+ lymphocytes increased in the brains on Day 6 p.i. The elimination of CD3+ lymphocytes or CD4+ lymphocytes augmented viral replication and suppressed the production of IFN-γ. The suppression of IFN-γ production by anti-CD3 monoclonal antibody (mAb) persisted, although the suppression by anti-CD4 mAb was observed only on Day 6 p.i. The depletion of CD8+ lymphocytes as well as TCR-αβ+ lymphocytes also augmented the viral replication; however, it did not alter the production of IFN-γ. Anti-Asialo GM1 antibody had no effect on viral replication and IFN-γ production. These results indicate that T lymphocytes are important for eliminating Theilers virus from the brain, CD3+/CD4+/CD8− lymphocytes and CD3+/TCRαβ−/CD4−/CD8− lymphocytes would produce IFN-γ in brain. However, from the result on the experiment of the depletion of TCR-αβ+ lymphocytes, the defence mechanisms by T lymphocytes against Theilers virus would be independent of endogenous IFN-γ production.

PARALYSIS CAUSED BY ACUTE MYELITIS IN THEILER'S MURINE ENCEPHALOMYELITIS VIRUS STRAIN GD VII INFECTION IS INDUCED BY CD4+ LYMPHOCYTES INFILTRATING THE SPINAL CORD

Intravenous infection by Theilers murine encephalomyelitis virus strain GD VII causes acute encephalomyelitis and paralysis in infected mice. However, nude mice and cyclophosphamide-treated ddY mice did not show paralysis when they were able to survive until day 20 post-infection (p.i.). Of ddY mice infected with 5 x 10(7) p.f.u./mouse, 70-80% showed symptoms of paralysis on day 20 p.i. The viral titres in the brain and spinal cord in infected mice were not significantly different between paralytic and non-paralytic mice. In all of the mice infected with the virus, CD4+ lymphocytes and CD8+ lymphocytes had infiltrated the brain on days 10, 12, 14 and 20 p.i. as demonstrated by flow cytometric analysis. In contrast, few T lymphocytes infiltrated the spinal cord in the non-paralytic mice. Administration of an anti-CD4 monoclonal antibody (MAb) or anti-T cell receptor-alpha beta MAb on day 6 p.i. inhibited paralysis until day 20 p.i., though 20% of the MAb-treated mice and 80% of the control mice showed paralysis. Administration of anti-CD8 MAb was not effective in the suppression of paralysis. The MAb treatment did not significantly augment viral replication in the spinal cord, although the viral titres in the brain of the MAb-treated mice increased significantly. After the transfer of spleen cells from infected C3H mice, the recipient mice infected with a small amount of the virus showed paralysis, though uninfected mice did not. This transfer could be blocked by CD4+ lymphocyte depletion of the donor mice. These results indicate that paralysis caused by acute myelitis in Theilers virus strain GD VII infection is induced by CD4+ lymphocytes infiltrating the spinal cord.

CD3 + /TCR-αβ - Cells Are Important in Protecting Spinal Cord Tissues against Theiler's Virus Strain GD VII Infection

Intravenous infection with Theilers virus strain GD VII causes acute encephalomyelitis in mice. Endogenous IFN‐γ produced in the spinal cord is important to protect the tissue in mice infected with this virus. Neither CD4+ cells nor CD8+ cells infiltrated the spinal cords of infected mice until Day 9 postinfection. However, the number of CD3+/TCR‐γδ+ cells increased in the spinal cords of mice infected with the virus. These cells resided in the spinal cords of normal mice, and produced IFN‐γ as a result of stimulation by immobilized anti‐CD3 mAb. Elimination of CD3+ cells by the administration of a specific mAb augmented viral replication and suppressed production of endogenous IFN‐γ. Depletion of TCR‐αβ+ cells and ASGM1+ cells did not affect the viral replication, and did not alter the production of IFN‐γ. Therefore, CD3+/TCR‐αβ– cells producing IFN‐γ play an important role in the protection of the spinal cord against Theilers virus infection. These results suggest that CD3+/TCR‐αβ– cells might be identical to TCR‐γδ+ cells.