J Toxicol Pathol 7: 371•`377, 1994 THE RELATIONSHIP BETWEEN INCREASED EMPERIPOLESIS INDEX AND SEVERE HEMATOPOIETIC CELL HYPERPLASIA IN THE BONE MARROW OF LPS-TREATED RATS Masaharu Tanaka, Yoshiya Aze, and Tsuneo Fujita FukuiInstitute for SafetyResearch, ONO Pharmaceutial Co., Ltd. Abstract: To investigate the relationship between the frequency of megakaryocytic emperipolesis and the number of hematopoietic cells, the animals were administered lipopolysaccharide (LPS) intravenously at a daily dose of 0.5mg/kg body weight for up to 7days, and histopathology of bone marrow and hematology of peripheral bloods were examined on Hour 2, 8, and 24 and Day 3, 5, and 7 of LPS treatment. Mature neutrophils appeared to be the most common hematocytes engulfed by megakar yocytes. Engulfed blood cells were observed in the demarcation membrane system of mature megakar yocytes. Cell membranes of both megakaryocytes and entering cells were intact. Although megakar yocytic emperipolesis was also found in control rats, its incidence increased markedly in LPS-treated rats which showed hyperplasia of granulocytes and megakaryocytes in their bone marrows. The index of emperipolesis showed a minimal change from 2.25 to 3.04 during the experimental period in control rats, whereas it rose from Hour 8 (9.78) and peaked on Day 3 (46.04) in LPS-treated rats. Hyperplasia of hematopoietic cells such as megakaryocytes and segmented neutrophils also peaked on Day 3 of LPS treatment. These changes suggest that emperipolesis is a phenomenon closely related to severe hematopoietic hyperplasia in the bone marrow. (J Toxicol Pathol 7: 371•`377, 1994) Key words: Emperipolesis, Lipoplolysaccharide, Megakaryocyte, Neutrophil, Rat normal bone marrows, it showed a marked Introduction increase under several pathological conditions; Emperipolesis was introduced by Humble et idiopathic thrombocytopenic purpura4,17, iron al to describe "inside round about wandering" of deficiency anemia 17, malignant tumors 1.4,10,12-18, lymphocytes within malignant cells in tissue etc. Under experimental conditions such as blood culture1. This phenomenon differs from loss5 and myelogenous leukemia7, there are some phagocytosis because entering cells temporary exist renorts of emnerinolesis in the rat bone marrow. within a host cell1-3. In addition, such structural In a previous study, we observed megakar characteristics of phagocytosis as formation of yocytes containing mature neutrophils in the rat endocytic vesicles and/or phagosomes are not seen bone marrow by 4-week LPS-administration. In in ultrastructural observation4-6. Host cells were that study, an increase in the incidence of megakar usually megakaryocytes17-11, and entering cells yocytic emperipolesis and the number of megakar were neutrophils4,6.9,11,12 lymphocytes1.6.9.10, and yocytes was detected19. erythrocytes5,8,9. Although megakaryocytic In the present study, we evaluated the relation emperipolesis has also been recognized in the ship between the frequency of megakaryocytic emperipolesis and the number of hematopoietic 田中雅治 阿瀬善也 藤田常夫 cells in the bone marrow of the LPS-treated rats. Accepted for publication: May 28 1994 Mailing address: Masaharu Tanaka, Fukui Institute for Safety Research, Ono Pharmaceutical Co., Ltd., 50-10 Yamagishi, Mikuni-cho, Sakai-gun, Fukui 913, Japan. 372 MEGAKARYOCYTIC EMPERIPOLESIS IN LPS-TREATED RATS For electron microscopic study, the right Materials and Methods femoral marrow was fixed in 3% glutaraldehyde in 0.1M phosphate buffer (pH 7.4) and then in 1% Animals osmium tetroxide in the same buffer. The tissues Sixty 6-week-old male rats of the Sprague were dehydrated in graded alcohol, embedded in Dawley strain were purchased from Clea Inc., Epon 812, sectioned by ultramicrotome (LKB Osaka, Japan. Animals were placed in hanging Ultrotome V, LKB Produkter AB, Stockholm, stainless wire cages (5 animals per cage) in a Sweden), and double-stained with uranyl acetate barrier system maintained at a temperature of 23•} and lead citrate. Ultra-thin sections were obser 2•Ž and a humidity of 55•}10% with 12 hours of ved using a transmission electron microscope light and darkness (from 8:00 to 20:00). They (JEM-100S, JEOL, LTD, Tokyo). were fed a standard pellet diet (CE-2, CLEA Emperipolesis index Osaka, Japan) and tap water ad libitum. After one week quarantine, they were used in this experi The number of megakaryocytes containing ment. segmented neutrophils (mature-type neutrophils) among a total of 1000 megakaryocytes were count Chemicals and experimental design ed on each H-E specimen. The number of mature Lipopolysaccharide (LPS) from Salmonella type neutrophils were also counted for each mega abortus equi was obtained from SIGMA Chemical karyocyte. From these informations, emper Co., U.S.A. (Lot 14F4011). Thirty rats were ad ipolesis index was calculated. Emperipolesis ministered LPS intravenously at a daily dose of 0.5 index was defined as the incidence of megakar mg/5ml/kg body weight for up to 7days. The yocytes engaged in emperipolesis multiplied by the remaining 30rats received 5ml/kg body weight of average number of entering cells within the cyto saline in the same way and used as controls. Five plasm of megakaryocytes in each sample. animals of each group were killed at 2, 8, and 24 The number of hematopoietic cells hours after the first injection and on the 3rd, 5th, and 7th day of LPS-treatment, respectively. The numbers of megakaryocytes and mature type neutrophils were counted in the bone marrow Hematological examination with a light microscope. The number of megakar Blood samples were collected from the jugular yocytes was counted at •~200 magnification in vein of each animal under ether anesthsia, and the randomly selected ten fields and that of mature numbers of total white blood cells (WBC) and neutrophils was counted at •~1,600 magnification platelets were counted using an automatic blood in randomly selected twenty fields per animal. cell counter (MICROCELLCOUNTER CC Statistical analysis 180A, TOA Medical Electronics Co., LTD, Kobe). The number of segmented neutrophils was calcu Data of the emperipolesis incidence were lated using a blood cell analyzer (MICROX HEG analyzed for statistical significance with chi-squar 120A, OMRON TATEISHI Electronics Co., LTD, ed test, and other data were analyzed with Tokyo) on blood smears stained with Student's t-test. May-Giemsa. Histological examination of the bone marrow Results For light microscopic study, the left femoral Hematological findings (Table 1) bone including bone marrow was removed from each rat and fixed in 10% neutral buffered forma The number of WBC showed a transient lin. Following decalcification, the tissues were decrease on Hour 2 (26.0•~102/mm3) of LPS-treat embedded in paraffin, sectioned and stained with ment. The number of platelets showed a rapid hematoxyline and eosin (HE). decrease from on Hour 2 (103.8•~104/mm3) and Tanaka, Aze, Fujita 373 Table 1. Hematology of LPS-treated Rats 5 animals in each group were used for examination. Each value is expressed as mean•}S.D. WBC: white blood cell, Segmented N: segmented neutrophil, Plt: platelet Statistical significance was analyzed using Student's t-test (*p<0.05, **p<0.01, ***p<0.001). reached a minimum value on Day 3 (25.5•~104/ cytoplasmic debris of engulfed cells (Fig. 4). mm3) of LPS-treatment. This change tended to Electron microscopically, most of the host reverse on Day 5 (110.2•~104/mm3). The number cells showing emperipolesis were mature megakar of segmented neutrophils showed a rapid increase yocytes with demarcation membrane system and on Hour 8 (64.2•~102/mm3) and Hour 24 (63.7•~ platelet granules. Engulfed hematocytes were 102/mm3), and thereafter it showed a transient present within the demarcation membrane system decrease on Day 3 (6.8•~102/mm3) of LPS-treat of host cells (Fig. 5). The membrane of megakar ment. yocytes neither fused with that of engulfed hematocytes nor formed phagosomes. The cell Histological findings of the bone marrow membranes of both megakaryocytes and entering Light microscopically, megakaryocytic emper cells were intact (Fig. 6). ipolesis in the bone marrow was observed in both Emperipolesis index (Table 2) control and LPS-treated groups. The frequency of emperipolesis and the number of entering In control rats, the index of emperipolesis hematocytes per megakaryocyte, however, in changed from 2.25 to 3.04 during the experimental creased significantly in the LPS-treated rats that period. On the other hand, in the LPS-treated showed hvnerDlasia of nranulocvtes and meeakar rats, it rose markedly from on Hour 8 (9.78) and yocytes in their bone marrows, and megakar peaked on Day 3 (46.04) of LPS-treatment. This yocytes showing emperipolesis were enlarged in tendancy continued to the end of the experiment. the LPS-treated rats (Figs. 1 and 2). Most of the In addition, the incidence of emperipolesis hematocytes within cytoplasm of megakaryocytes (16.68%) and the number of mature neutrophils were mature neutrophils, though lymphocytes and within a megakaryocyte (2.76 cells) peaked on Day erythrocytes were also engulfed by megakar 3 of LPS-treatment. yocytes. Entering hematocytes were demarcated The number of hematopoietic cells in the bone from the cytoplasm of megakaryocytes by cystic marrow (Table 2) spaces (Fig. 3). Both entering cells and megakar yocytes generally appeared normal, but megakar The number of megakaryocytes in the bone yocytes occasionally contained nuclear and/or marrow of the LPS-treated rats began to increase