Mariko Akiyama

Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice.

Chronic intermittent injection of carbon tetrachloride (CCI4) for more than 10 weeks induced liver fibrosis in mice, as evidenced by positive Azan staining and increased intrahepatic collagen content. Preceding the onset of liver fibrosis, interleukin‐6 (IL‐6) gene expression was enhanced in liver and immunoreactive IL‐6 was detected in infiltrating inflammatory cells. To delineate the role of IL‐6 in this process, we treated IL‐6‐deficient mice with CCl4 in a similar manner for 12 weeks, after which fibrotic changes were less evident and serum albumin levels were lower in IL‐6‐deficient than wild‐type mice. Moreover, CCl4‐induced expression of transforming growth factor β1 and hepatocyte growth factor genes in liver was significantly reduced in IL‐6‐deficient mice. Thus, IL‐6 may be vitally involved in fibrotic changes and maintenance of serum albumin levels, partly by modulating intrahepatic expression of these cytokines. J. Leukoc. Biol. 66: 601–608; 1999.

Preparation of specific antibodies against murine IL-1ra and the establishment of IL-1ra as an endogenous regulator of bacteria-induced fulminant hepatitis in mice.

Blocking monoclonal antibodies (mAbs) specific to mouse interleukin‐1 receptor antagonist (IL‐1ra) were prepared by immunizing Armenian hamsters with recombinant mouse IL‐1ra. A sensitive and specific ELISA against mouse IL‐1ra was also established. In Propionibacterium αcnes‐induced liver injury, P. acnes induced transient increase of serum tumor necrosis factor‐α levels but not those of IL‐1ra, IL‐1, and IL‐6. However, subsequent lipopolysaccharide (LPS) challenge induced the increase of serum levels of all these cytokines and the peak serum IL‐1ra level was more than 20 times as high as serum IL‐1 levels. Immunohistochemical analysis demonstrated that IL‐1ra was predominantly produced by hepatocytes during the course of the priming phase by P. acnes and eliciting phase by LPS challenge. Furthermore, the administration of a mAb to mouse IL‐1ra exacerbates the liver injury induced by P. acnes and sublethal dose of LPS, suggesting a protective role of endogenous IL‐1ra in this liver injury model. J. Leukoc. Biol. 58: 90–98; 1995.

Pathogenic roles of tumor necrosis factor receptor p55-mediated signals in dimethylnitrosamine-induced murine liver fibrosis.

TNF-α has pleiotropic functions, but its role in liver fibrosis has not yet been clarified. To understand the pathophysiologic role of the TNF-α/TNF receptor (TNFR) p55 signals in liver fibrosis, 10 mg/kg of dimethylnitrosamine, a specific hepatotoxicant, was administered twice a week into the peritoneal cavity of both TNFRp55 knock-out (KO) and wild-type mice, and the severity of fibrosis was monitored histologically and biochemically. In wild-type mice, histologic analysis demonstrated evident fibrotic changes 1 week after the initiation of dimethylnitrosamine administration, consistent with increased liver collagen contents. Concomitantly, the numbers of Kupffer cells and activated hepatic stellate cells (HSCs) were increased in liver tissue. On the contrary, fibrotic changes were attenuated and the numbers of Kupffer cells and HSCs were decreased in TNFRp55-KO mice. Moreover, gene expression of TNF-α and monocyte chemoattractant protein-1, which are involved in Kupffer cell activation or migration, was decreased in the liver of TNFRp55-KO mice. Collectively, TNFRp55-mediated signals may regulate activation of Kupffer cells and HSCs and eventually enhance fibrotic process.

Isolation of a novel retina-specific clone (MEKA cDNA) encoding a photoreceptor soluble protein

We have reported the isolation of clones which are candidates for retina-specific cDNAs. One of the cDNA clones, pCR-470, was further characterized. We found that mRNA corresponding to the pCR-470 was expressed only in the retina and encodes an unknown soluble protein whose molecular weight and pI are calculated to be 26,935 and 5.35, respectively. We designated it as a MEKA protein, because its amino acid sequence starts from M-E-K-A. It was found by in situ hybridization that MEKA mRNA was transcribed only in the photoreceptor cells and accumulated in the inner segments just like opsin mRNA. The MEKA cDNA was ligated with expression vector PEX 1, and a MEKA-fusion protein synthesized in E. coli was purified and used as an antigen. By the Western blot analysis anti-MEKA protein serum reacted with a soluble 32 kDa protein from bovine retina and 33 kDa for chick, but not with proteins from other tissues. Immunohistochemical study showed that anti-MEKA stained only the photoreceptor cells in bovine, chick, rat and mouse retinas.

Presence of retina-specific proteins in the lamprey pineal complex.

The pineal complex of river lamprey reacted with the antisera raised against retina specific proteins including bovine opsin, chick visinin and frog light-sensitive cyclic GMP phosphodiesterase (PDE). Immunoreactive materials stained with anti-opsin were evenly located at the outer segment of photoreceptor cells in the pineal organ and also found in the parapineal organ. Although anti-visinin stained the pineal and parapineal photoreceptor cells, the immunopositive photoreceptor cells were observed only at the lateral portion and not at the medial portion of the pineal organ. No immunoreactive materials were found in the pineal complex by the anti-PDE, whereas the anti-PDE reacted with photoreceptor cells of the retinal tissue. The data suggest that the pineal and parapineal retinas of lamprey contain opsin- and visinin-like proteins with different distribution in their photoreceptor cell layer as found in the lamprey retinal tissue.

Potential involvement of monocyte chemoattractant protein (MCP)-1/CCL2 in IL-4-mediated tumor immunity through inducing dendritic cell migration into the draining lymph nodes

We previously observed that IL-4 gene transduction into a mouse colon 26 adenocarcinoma cell line abrogated its tumorigenicity due to the generation of anti-tumor CTL. DEC-205- and CD11c-double positive cells were increased in the lymph nodes of mice injected with IL-4-transfected cells between 2 and 3 days after the tumor injection, compared with those injected with parental cells. Most of these double positive cells expressed CD86 antigen. Among the chemokines with chemotactic activities against dendritic cells, monocyte chemoattractant protein (MCP)-1/CCL2, ABCD-1/CCL22, and liver and activation-regulated chemokine (LARC)/CCL20 gene expression was enhanced no later than 3 days after the tumor injection, in the draining lymph nodes of IL-4-transfected cell bearing mice. Moreover, gene expression of the receptor for MCP-1/CCL2, CCR2, was enhanced in the draining lymph nodes of the mice injected with IL-4-transfected cells, and most DEC-205-positive cells in the lymph nodes expressed CCR2. Finally, the administration of anti-MCP-1/CCL2 antibodies retarded the rate of tumor regression in mice injected with IL-4-tranfected cells, concomitantly with a decrease in DEC-205- and CD11c-double positive cell number in the draining lymph nodes. Thus, locally produced MCP-1/CCL2 may be responsible for IL-4-mediated tumor rejection presumably based on the induction of dendritic cell migration into the draining lymph nodes.

Participation of Endogenously Produced Interferon gamma in Interleukin 4-Mediated Tumor Rejection

To elucidate the molecular mechanism underlying IL-4-induced tumor rejection, we challenged mice with a mouse adenocarcinoma cell line, colon 26, genetically engineered to express constitutively IL-4 gene (colon 26/IL-4). Immunocompetent BALB/c mice rejected colon 26/IL-4 cells but not parental cells or cells transduced with a control gene (colon 26/control). Moreover, on rechallenge, parental cells and colon 26/control cells were rejected by normal BALB/c mice that had previously rejected colon 26/IL-4. However, both nude and severe combined immunodeficiency (SCID) mice failed to reject colon 26/IL-4 as well as parental or colon 26/control cells. In contrast, nude mice did reject colon 26/IL-4 after transfer of lymphocytes obtained from the draining lymph nodes of BALB/c mice injected with colon 26/IL-4. These results indicate that challenging mice with colon 26/IL-4 tumor cells resulted in the generation of memory cytotoxic T lymphocytes in the draining lymph nodes. At 3 days after the challenge, IFN-gamma, IL-12 p35, and p40 mRNA expression was selectively enhanced in the draining lymph nodes of mice bearing colon 26/IL-4 cells. Finally, mice deficient in the IFN-gamma gene did not reject colon 26/IL-4 cells. These results suggest that IL-4-induced memory cytotoxic T lymphocyte generation requires IFN-gamma production in the draining lymph nodes, in order to generate a protective immune response.

FUNCTIONAL ROLE OF CALCIUM IN PHOTORECEPTOR CELLS

Abstract— Ca‐uptake by disc membranes prepared from frog rod photoreceptor outer segments was examined. Ca‐uptake study revealed two affinity sites which were saturated with 10–5M and 10–3M of ATP. When disc membranes in 20 mM Tris‐HCl buffer (pH 7.5) were stored at ‐20°C for 6 h, more than 95% of Ca‐uptake activity was lost. Ca‐uptake activity was, however, preserved if the disc membrane suspension was mixed with 1–10mM ATP and stored at ‐20°C. Furthermore the reactivation of Ca‐uptake was observed if disc membranes, which had lost Ca‐uptake ability by storing at 4°C for 3 h, were mixed with 10 mM ATP and then frozen at ‐20°C for 5 h or 28 h (ATP‐induced ATP‐dependent Ca‐uptake). When the contents of ATP bound to disc membranes were measured during a brief aging at 37°C, the decrement of bound ATP content was correlated well with the decreasing of Ca‐uptake activity. Carbonylcyanide m‐chlorophenylhydrazone (CCCP), a conductor of protons, inhibited Ca‐uptake activity and half maximal inhibition was achieved at 2 × 10–8M. When 10–6M of CCCP was added to the 45Ca‐accumulated disc membranes, rapid release of 45Ca from the disc membranes was observed. These results suggest that ATP may play a role in the Ca‐pump regulation in disc membranes and a [H+] gradient across disc membrane may be linked to Ca‐uptake mechanism.

Ouabain binding to chick embryo neuroretina during development in ovo and in monolayer cultures

Eight-day-old embryo neuroretinas (NR) were dissociated and cultured as monolayer. [3H]Ouabain binding was investigated during the culture period. [3H]Ouabain binding to NR increases continuously until around the tenth day of culture, but after that it gradually decreased. On the other hand, [3H]ouabain binding to NR during in ovo development continued to increase until the embryo was 20 days old. Na-K-ATPase activity was also compared with [3H]ouabain binding during development in ovo and in monolayer culture. It was found that changes in Na-K-ATPase activity of NR in vitro and in ovo were different from those of [3H]ouabain binding activity. Exposure of NR at the second day of culture to 0.1 mM ouabain for 8 h resulted in the degeneration of some differentiated neurons (rosette-formed cells), but new rosettes were soon re-formed after the withdrawal of ouabain, and [3H]ouabain binding to the ouabain-treated NR was decreased by 30%. Ouabain-treatment of NR in 10-day-old cultures completely destroyed the aggregated neurons, but did not apparently injure the glial cell layer. [3H]Ouabain binding decreased more than 90% in the ouabain-treated NR. These results suggest that [3H]ouabain binding activity of NR and the sensitivity to ouabain toxicity are useful indicators for differentiation and development of neurons in the cultured retinal cells.

Differentiation of neuroblastoma cells by chick gizzard extract

Cholinergic neuroblastoma NS20Y cells were differentiated by the chicken gizzard extract. They were first inoculated into a glass culture bottle and the aggregated cells which grew in the suspension culture were collected. The aggregated cells (round and immature neuroblastoma cells) were seeded on a polyornithinecoated plastic dish, and the effect of various agents on the differentiation of the neuroblastoma was investigated. When gizzard extract from chicken was added to the culture, many flat cells with neurites emerged around the cell aggregates within 24 h. The flat cells could evoke action potentials with high frequency (in 70% cells). Cyclic GMP levels in the treated cultures were much lower than that in the control culture, and remained continuously lower during 2 days culture. The factor responsible for the differentiation of neuroblastoma cells was rich in the chick gizzard among extracts or conditioned media from various tissues tested. A similar effect was observed by the addition of dibutyryl cyclic AMP or prostaglandin E(1) plus theophylline over a slower time course. The factor in gizzard extract was trypsin-sensitive and heat-labile. The molecular size was estimated to be about 12 s.