Keiko Watano

Allograft inflammatory factor-1 augments production of interleukin-6, -10and -12 by a mouse macrophage line

Mouse allograft inflammatory factor‐1 (AIF‐1) cDNA was cloned and the AIF‐1‐specific monoclonal antibodies were established to examine its tissue distribution. The mouse AIF‐1 was highly conserved among all reported AIF‐1 from a variety of species, from invertebrates to mammals, and the cloned cDNA was in good accordance with putative expressed regions of genomic sequences in the mouse major histocompatibility complex (MHC) class III region. The messages of mouse AIF‐1 were abundantly expressed in the testis, moderately in the spleen and lymph nodes and slightly in the liver and thymus of normal BALB/c mice. Immunohistological examination revealed that differentiating germ cells in the testis and presumably macrophages in the red pulp of the spleen were positive for AIF‐1. To analyse the function of the AIF‐1, a macrophage cell line, RAW 264.7, was transfected with mouse AIF‐1 cDNA. Upon stimulation with bacterial lipopolysaccharide, the transfectants that overexpressed AIF‐1 showed marked morphological changes and produced significantly large amounts of interleukin (IL)‐6, IL‐10 and IL‐12p40 but not IL‐12p70 compared with control cells. No difference was noted in production of tumour necrosis factor‐α, transforming growth factor‐β1 and IL‐1α. These results suggest that AIF‐1 plays an important role in cells of a monocyte/macrophage lineage upon stimulation with inflammatory stimuli by augmenting particular cytokine production.

Angiotensin-Converting Enzyme Inhibition Attenuates Hypofibrinolysis and Reduces Cardiac Perivascular Fibrosis in Genetically Obese Diabetic Mice

Background—Obesity and insulin resistance are associated with accelerated macrovascular and microvascular coronary disease, cardiomyopathic phenomena, and increased concentrations and activity in blood of plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of fibrinolysis. Methods and Results—To determine whether hypofibrinolysis in blood and tissues and its potential sequelae could be attenuated pharmacologically, we studied genetically modified obese mice. By 10 weeks of age, obese mice exhibited increases in left ventricular weight and glucose and immunoreactive insulin in blood. PAI-1 activity in blood measured spectrophotometrically was significantly elevated as well. The difference compared with values in lean controls widened by 20 weeks of age. Perivascular fibrosis in coronary arterioles and small coronary arteries was evident in obese mice 10 and 20 weeks of age, paralleling increases in PAI-1 and tissue factor expression evident by immunohistochemical image analysis, in situ hybridization, and reverse transcription-polymerase chain reaction. Inhibition of ACE activity initiated in obese mice 10 weeks of age and continued for 20 weeks arrested the increase in PAI-1 activity in blood and in cardiac PAI-1 and tissue factor mRNA as well as coronary perivascular fibrosis. Conclusions—Thus, inhibition of proteo(fibrino)lysis and augmented tissue factor expression in the heart precede and may contribute to the coronary perivascular fibrosis seen with obesity and insulin resistance. Furthermore, inhibition of ACE activity can attenuate all 3 phenomena.

Expression of allograft inflammatory factor-1 in mouse uterus and poly(I:C)-induced fetal resorption.

Problem: To investigate whether the allograft inflammatory factor‐1 (AIF‐1) is expressed and plays a role in the reproductive system.

Induction of Plasminogen Activator Inhibitor-1 in Endothelial Cells by Basic Fibroblast Growth Factor and Its Modulation by Fibric Acid

Plasminogen activator inhibitor-1 (PAI-1) inhibits fibrinolysis and proteolysis. Basic fibroblast growth factor (bFGF) stimulates angiogenesis, which requires regional proteolysis. Because modulation of vasculopathy requires tight control of proteolysis, effects of bFGF on PAI-1 expression in endothelial cells (ECs) were characterized. bFGF increased PAI-1 mRNA and accumulation of PAI-1 protein in conditioned media in human umbilical vein ECs. The bFGF-mediated increase in PAI-1 mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. The rate of decrease in PAI-1 mRNA after actinomycin D treatment was not affected by bFGF. Transient transfection assays of the human PAI-1 promoter-luciferase construct demonstrated that bFGF-induced PAI-1 transcription was dependent on the elements within the −313 to −260 bp relative to the transcription start site. This region contains an E26 transformation specific 1 (Ets-1)-like site. Electrophoretic mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of the Ets-1-like transcription factor to the PAI-1 promoter. Nucleotide substitution to disrupt the Ets-1-like site reduced bFGF-stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator-activated receptor-&agr;, inhibited basal and bFGF-stimulated PAI-1 expression. By inducing PAI-1 expression from ECs, bFGF may control proteolysis and fibrinolysis in vessel walls.

Allograft inflammatory factor-1 regulates trinitrobenzene sulphonic acid-induced colitis

The expression of allograft inflammatory factor‐1 (AIF‐1) in 2,4,6‐trinitrobenzene sulphonic acid (TNBS)‐induced colitis, a model for T helper 1 (Th1) type disease, was investigated in BALB/c mice. The AIF‐1 expression was significantly increased in the colitis lesion compared to that in the normal colon. We then prepared AIF‐1 transgenic mice (Tgm) with the BALB/c background that express high levels of AIF‐1 in lymphoid tissues and the colon. When AIF‐1 Tgm were administrated TNBS, the TNBS‐induced colitis was ameliorated compared with that in non‐transgenic littermates. The amelioration of colitis was associated with the low expression of interleukin‐1β in the colon. The present findings suggest that AIF‐1 regulates Th1‐type inflammatory responses.

Delayed Clearance of Zymosan-induced Granuloma and Depressed Phagocytosis of Macrophages with Concomitant Up-regulated Kinase Activities of Src-family in a Human Monocyte ChemoaHractant Protein-1 Transgenic Mouse

A human monocyte chemoattractant protein-1 (hMCP-1) transgenic mouse (Tgm) line which constitutively produces a large amount of hMCP-1 (7-13 ng/ml in the serum) was established. Although expression of the transgene was detected in various tissues, an accumulation of macrophages (Mphi) was seen in only lymphoid organs which might be attributed to the high concentration of hMCP-1 in these organs. A reduced phagocytosis by peritoneal Mphi in vivo and a delayed clearance of granulomas in the liver following zymosan administration were observed in these Tgm. However, peritoneal exudate cells (PEC) from Tgm exhibited normal in vitro phagocytic activity and nitric oxide (NO) production upon stimulation with IFN-gamma as compared with those from non-Tgm. In addition, high activities of src-family protein tyrosine kinases (PTK), Fgr and Hck, were also noted in the peritoneal resident cells from Tgm, whereas the level of mitogen-activated protein kinase (MAPK) activity was almost the same as that of non-Tgm. It was suggested that the low functional activities of Tgm Mphi seen in vivo were attributed to down-regulation of the unique transducing system of hMCP-1 signals under the influence of a high concentration of the hMCP-1. It seemed that the depressed functions were recovered when the peritoneal cells were released ex vivo from such a high hMCP-1 environment.

Mixed allogeneic chimerism with wild-type strains ameliorates atherosclerosis in apolipoprotein E-deficient mice

Atherosclerosis involves inflammatory processes between vasculartissues and hematocytes with a hyperlipidemic background. To examinewhether variations of hematocytes constitute one of the geneticcomponents in atherosclerosis, irradiated apolipoprotein E(apoE)‐deficient (apoE−/−) mice with hypercholesterolemiaand preexisting atherosclerotic lesions were reconstituted withmixed bone marrow cells (BMC) from syngeneic and wild‐type(apoE+/+; atherosclerosis‐resistant SJL or ‐susceptibleB10.S) mice. Stable mixed allogeneic chimeras with small amounts ofserum apoE were established without any detrimentalcomplications. Compared with untreated apoE−/− miceor apoE−/− mice transplanted with syngeneic BMC alone, significant reduction of the cholesterol level and significant lesionregression were observed in the mixed chimeras. Furthermore, mixedchimeras given SJL BMC showed marked reductions in numbers of lesionscompared with those reconstituted with B10.S BMC. Cholesterol levels inthe former SJL chimeras, however, were significantly higher than thosein the latter B10.S chimeras. These findings indicate that theresistance of SJL to atherosclerosis resides in the bone marrow‐derivedcells.