Toru Hara

Abolition of anti-glomerular basement membrane antibody-mediated glomerulonephritis in FcRγ-deficient mice

Several recent studies have demonstrated the central role of Fc receptors (FcR) rather than the complement system in triggering hypersensitivity reactions. We investigated the role of FcR for IgG (FcγR) using a murine model of accelerated anti‐glomerular basement membrane (GBM) antibody‐mediated glomerulonephritis as a representative of type II hypersensitivity diseases. Intravenous injection of rabbit anti‐GBM antibody after preimmunization with normal rabbit IgG induced proteinuria and azotemia in wild‐type C57BL / 6 and CD40+ / – mice but not in FcR γ chain (FcRγ)– / – mice or CD40– / – mice. Light microscopic findings revealed marked tissue damage in the glomeruli of wild‐type C57BL / 6 and CD40+ / – mice. However, no tissue damage except polymorphonuclear cell infiltration was observed in the glomeruli of FcRγ– / – mice. The glomeruli of CD40– / – mice were almost normal. Immunohistochemistry revealed the binding of rabbit IgG to the GBM in all mice injected with anti‐GBM antibody. However, depositions of mouse IgG and complement to the glomeruli were not observed in CD40– / – mice, and deposition of fibrin was not observed in FcRγ– / – or CD40– / – mice. These findings suggest that FcγR may initiate anti‐GBM antibody‐mediated renal disease. We conclude that FcγR rather than the complement system is critically involved in the development of type II hypersensitivity diseases.

Induction of antitumor immunity by transduction of CD40 ligand gene and interferon-gamma gene into lung cancer.

CD40–CD40 ligand (CD40L) interaction is an important costimulatory signaling pathway in the crosstalk between T cells and antigen-presenting cells. This receptor–ligand system is known to be essential in eliciting strong cellular immunity. Here we demonstrate that murine lung cancer cells (3LLSA) transduced with the CD40L gene (3LLSA-CD40L) were rejected in syngeneic C57BL/6 mice, but grew in CD40-deficient mice to the same extent as control tumor cells. Immunohistochemical study showed that inflammatory cells, including CD4+, CD8+ T cells and NK cells, infiltrated into the inoculated 3LLSA-CD40L tumor tissue. Inoculation of 3LLSA-CD40L cells into mice resulted in the induction of 3LLSA-specific cytotoxic T-cell immunity, and the growth of parental 3LLSA tumors was inhibited when 3LLSA cells were inoculated into C57BL/6 mice mixed with 3LLSA-CD40L cells or when they were rechallenged 4 weeks after 3LLSA-CD40L cells were rejected. Furthermore, co-inoculation of interferon (IFN)-γ–transduced cells (3LLSA-IFNγ) with 3LLSA-CD40L cells enhanced the antitumor immunity efficiently in vivo. These results indicate that the in vivo priming with CD40L- and IFN-γ gene–transduced lung cancer cells is a promising strategy for inducing antitumor immunity in the treatment of lung cancer. Cancer Gene Therapy (2001) 8, 421–429

Effect of Gene Transfer of Tumor Necrosis Factor Receptors into Human Lung Carcinoma Cell Line

The human lung adenocarcinoma cell line A549 is known to be resistant to tumor necrosis factor alpha (TNF‐α)‐mediated tumor cell lysis in spite of the expression of 55 kDa TNF receptor (TNF‐R55) mRNA and its cell surface protein. In this study, we investigated the mechanism of TNF‐α resistance and the role of two types of TNF receptors (TNF‐R55 and TNF‐R75 (75 kDa TNF receptor)). TNF‐R55 or TNF‐R75 cDNA was transfected into A549 cells. In addition, a C‐terminal deletion mutant of TNF‐R75 which lacks the intracellular domain of TNF‐R75 was also transfected into A549 cells. We assessed the TNF‐α‐mediated tumor cell lysis of these transfected clones, and found that the cytotoxic effect increased in transfected clones highly expressing TNF‐R55, but not in low‐expression clones. As for TNF‐R75, the cytotoxic effect of TNF‐α was observed in TNF‐R75‐transfected clones even when expression was low. Furthermore, the cytotoxic effect was also observed in clones transfected with the deletion mutant of TNF‐R75, as well as the complete TNF‐R75. These results indicate that a certain level of expression of TNF‐R55 is necessary for obtaining TNF‐α‐mediated tumor cell lysis in the absence of TNF‐R75. On the other hand, the expression of TNF‐R75 strongly induces TNF‐α‐mediated cytotoxicity through TNF‐R55 in the absence of an intracellular signal via TNF‐R75.

Thymus-dependent modulation of Ly49 inhibitory receptor expression on NK1.1+γ/δ T cells

Major histocompatibility complex (MHC) class I‐specific inhibitory receptors are expressed not only on natural killer (NK) cells but also on some subsets of T cells. We here show Ly49 expression on γ/δ T cells in the thymus and liver of β2‐microglobulin‐deficient (β2m−/−) and C57BL/6 (β2m+/+) mice. Ly49C/I or Ly49A receptor was expressed on NK1.1+γ/δ T cells but not on NK1.1−γ/δ T cells. The numbers of NK1.1+γ/δ T cells were significantly smaller in β2m+/+ mice than in β2m−/− mice with the same H‐2b genetic background. Among NK1.1+γ/δ T cells, the proportions of Ly49C/I+ cells but not of Ly49A+ cells, were decreased in β2m+/+ mice, suggesting that cognate interaction between Ly49C/I and H‐2Kb is involved in the reduction of the number of Ly49C/I+γ/δ T cells in β2m+/+ mice. The frequency of Ly49C/I+ cells in NK1.1+γ/δ T cells was lower in both lethally irradiated β2m+/+ mice transplanted with bone marrow (BM) from β2m−/− mice and lethally irradiated β2m−/− mice transplanted with BM from β2m+/+ mice than those in adult thymectomized BM‐transplanted chimera mice. These results suggest that reduction of Ly49C/I+ NK1.1+γ/δ T cells in β2m+/+ mice is at least partly due to the down‐modulation by MHC class I molecules on BM‐derived haematopoietic cells or radioresistant cells in the thymus.

Detection of loci for allergic asthma using SMXA recombinant inbred strains of mice

Asthma is regarded as a multifactorial inflammatory disorder arising as a result of inappropriate immune responses in genetically susceptible individuals to common environmental antigens. However, the precise molecular basis is unknown. To identify genes for susceptibility to three asthma-related traits, airway hyperresponsiveness (AHR), eosinophil infiltration, and allergen-specific serum IgE levels, we conducted a genetic analysis using SMXA recombinant inbred (RI) strains of mice. Quantitative trait locus analysis detected a significant locus for AHR on chromosome 17. For eosinophil infiltration, significant loci were detected on chromosomes 9 and 16. Although we could not detect any significant loci for allergen-specific serum IgE, analysis of consomic strains showed that chromosomes 17 and 19 carried genes that affected this trait. We detected genetic susceptibility loci that separately regulated the three asthma-related phenotypes. Our results suggested that different genetic mechanisms regulate these asthma-related phenotypes. Genetic analyses using murine RI and consomic strains enhance understanding of the molecular mechanisms of asthma in human.