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P036 Apoptotic neutrophils ameliorate intestinal inflammation - possible mechanism of granulocyte/monocyte apheresis

S. Ishihara1, K. Kawashima*1, H. Sonoyama1, Y. Tada1, A. Oka1, R. Kusunoki1, N. Fukuba1, N. Oshima1, I. Moriyama2, T. Yuki3, H. Maegawa4, N. Kashiwagi4, Y. Kinoshita1

1Shimane University Faculty of Medicine, Internal Medicine II, Izumo, Japan, 2Shimane University Hospital, Cancer center, Izumo, Japan, 3Shimane University Hospital, Gastrointestinal Endoscopy, Izumo, Japan, 4JIMRO Co., Ltd, Research division, Takasaki, Japan


Granulocyte/monocyte apheresis (GMA) is used as a therapeutic option for induction therapy for inflammatory bowel diseases (IBD). An Adacolumn is an adsorptive type carrier-based medical device for GMA and its major components are cellulose acetate beads. The main concept behind the development of the Adacolumn is removal of activated leukocytes. However, the actions of the column are more than just removing leucocytes, as a type of immunomodulation has also been suggested in Results of several clinical and basic research studies. For the present study, we hypothesized that apoptosis is induced in leukocytes by reactive oxygen species (ROS) generated in the Adacolumn, and a considerable number of apoptotic leukocytes re-enter the body and contribute to the efficacy of GMA.


A rabbit GMA model was established as reported previously (Inflammation. 2002). We used a mini GMA column with a diameter of 1.5 cm and length of 10 cm, which contained 11 g of cellulose diacetate carriers. The amount of O2- was measured by directly counting the number of photons emitted by MCLA upon reaction with O2-. Neutrophils were isolated from the outflow blood during GMA and apoptosis induction of those cells was evaluated by flow cytometry. Next, we established a SCID mouse colitis model by adoptive transfer of CD4+ T cells isolated from SAMP1/Yit mice, a murine model of Crohn's disease. Peritoneal exudate cells (PECs) were isolated for preparing injected apoptosis cells from a control strain AKR. Apoptosis was induced in PECs by H2O2 treatment and those apoptotic cells were intravenously injected to the colitis mice in the presence of co-transferred whole B cells. Seven weeks after colitis induction, the severity of colon inflammation was evaluated by weight loss, colon length, histopathology, and detection of inflammatory cytokines in colon tissues.


Photon counts were gradually increased after initiation of GMA, which was not found in a sham apheresis model. Generation of O2- in the column was confirmed by infusion of superoxide dismutase into the column. A significant decrease in L-selectin expression on neutrophils was observed in the GMA outflow, while hypodiploid apoptotic neutrophils were also significantly increased in the outflow. To mimic apoptotic neutrophils generated during GMA therapy, apoptosis was induced in PECs by exposure to H2O2. Intravenously injection of H2O2-induced neutrophils significantly reduced the colonic inflammation as compared to the PBS injection group.


Apoptosis was induced in circulating neutrophils by ROS generated in the Adacolumn, which may contribute to the anti-inflammatory effect of GMA as a novel therapeutic mechanism for IBD.