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 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 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, , , 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

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: , 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 374 MEGAKARYOCYTIC EMPERIPOLESIS IN LPS-TREATED RATS

after, it showed a transient increase on Day 3 (850.0 cells) in the LPS-treated rats as compared with that in control ones (659.2 cells).

Discussion

In this study, megakaryocytic emperipolesis was examined in the bone marrow of the LPS treated rats. As described before, megakaryocytic emperipolesis has been distinguished from phagocytosis by or neutrophils, because engulfed cells retain their normal struc tures in megakaryocytes1-3. Light microscopi cally, degeneration of engulfed cells within cyto

plasm of megakaryocytes has sometimes been found in the human and rat bone marrows4,11 The ultrastructual examination, however, revealed no changes both in engulfed hematocytes and megakaryocytes4-6. Except for mn occasional light microscopic finding of the existence of nuclear and/or cytoplasmic debris of engulfed cells in megakaryocytes, the light and electron micro scopic findings of megakaryocytic emperipolesis observed in the present study were almost similar to those sited above1-6,11. Namely, engulfed hematocytes were intactly seen within demarcation membrane systems of megakaryocytes with normal structures. Megakaryocytic emperipolesis has been repor

Figs. 2-6. The bone marrow of rats treated with LPS ted in humans under various clinical for 3 days. states1,4,10,12-16,18 and it was said to be associated Fig. 1. There are no remarkable changes in the bone with blood loss in some studies2.17,20 Endotoxin marrow. (Control, HE •~150) Fig. 2. Note an increase and enlargement of megakar (LPS) is well known to cause hematological and

yocytes as compared with Fig. 1. (LPS-treated histological alterations via direct and/or indirect group, HE •~150) effect21, and, in our previous studies, megakar Fig. 3. A megakaryocyte containing two neutrophils yocytic emperipolesis and peripheral anemic surrounded by a clear pericellular space (arrows). changes also observed in rats following LPS (LPS-treated group, HE •~600) Fig. 4. A megakaryocyte containing several hemato treatment19. cytes, mainly mature neutrophils. Note some Rozman and Vives-Corrons17 investigated the cellular debris-like materials (arrows). (LPS relationship between an increase in emperipolesis treated group, HE •~550) and number of megakaryocytes using bone marrow smears of total 365 patients and observed emper from on Hour 24 (235.2 cells), as compared with ipolesis and hyperplasia of megakaryocytes in 277 that of control ones (199.4 cells), and peaked on cases (77.8%). In rodents, Lee6 studied megakar Day 3 of LPS-treatment (346.8 cells). On the yocytic emperipolesis and reported an increase in other hand, the number of segmented neutrophils its incidence in the bone marrow of aged rats showed a rapid decrease on Hour 2 (328.8 cells) presenting granulocytic hyperplasia due to chronic and 8 (326.2 cells) in the LPS-treated rats. There blood loss and chronic inflammation. Tanaka, Aze, Fujita 375

Fig. 5. Note an entering leukocyte within the demarcation membrane system (arrow) of megakaryocyte.

(LPS-treated group, TEM •~3,700) Fig. 6. Note a neutrophil engulfed by a megakaryocyte. Entering cell and megakaryocyte show normal structure and no phagosome formation. (LPS-treated group, TEM •~3,700)

In this study, the index of megakaryocytic megakaryocytes, and segmented neutrophils, emperipolesis increased markedly following LPS peaked on Day 3 of LPS-treatment. This is con treatment in rats which showed hyperplasia of sidered to be a reaction against such hematological granulocytes and megakaryocytes in their bone changes as a clear decrease in number of peripheral marrows. The hyperplasia of hematopoietic cells, platelets and segmented neutrophils, because the 376 MEGAKARYOCYTIC EMPERIPOLESIS IN LPS-TREATED RATS

Table 2. Emperipolesis Index and Number of Hematopoietic Cells in the Bone Marrow of LPS-treated Rats

5 animals in each group were used. EP: emperipolesis EP-index=EP incidence (%)•~average No. of entering cells per megakaryocyte Statistical significance was analyzed using xz test (a), Student's t-test (b), *p<0.05, **p<0.01, ***p<0.001.

Fig. 7. Correlation of EP-index and the number of Fig. 8. Correlation of EP-index and the number of megakaryocytes. mature neutrophils.

alteration in the bone marrow has followed that in Several hypotheses of the cause of emper the peripheral blood. Furthermore, the index of ipolesis has been proposed. It is suggested that emperipolesis also peaked on Day 3 of LPS-treat emperipolesis is related to cellular traffic across the ment. Therefore, the index of emperipolesis marrow-blood barriers. Another author de showed a positive correlation with the numbers of scribed that an undefined insult to the bone mar megakaryocytes and mature neutrophils in the row, resulting in an increase in megakaryocyte bone marrow of the LPS-treated rats (Figs. 7 and turnover or death, might contribute to emper 8). ipolesis of neutrophils by megakaryocytes15. A Tanaka, Aze, Fujita 377 hypothesis that the cytoplasm of megakaryocyte an experimental myelogenous . Blood 39: 99-112, 1972. may provide a "sanctuary" for normal 8. Halil, O and Barrett, AJ: Phagocytosis by megakar granulocytes under an unfavorable bone marrow yocytes in malignant disorders. Br J Haematol 46: environment" is also proposed. Moreover, one 161. 1980. other hypothesis that emperipolesis is attributed 9. Breton-Gorius, J: On the alleged phagocytosis by megakaryocytes. Br J Haematol 47: 635-636, 1981. simply to a random movement and adherence of 10. Djaldetti, M and Strauss, Z: Emperipolesis by hematocytes to megakaryocytes20 is suspected. megakaryocytes in patients with non-Hogkin's Although the detail mechanism of emperipolesis is lymphoma and megaloblastic anemia. J Submi crosc Cytol 14: 407-413, 1982. still unknown, our results indicate that emper 11. Chiu, T: Megakaryocytes with intracytoplasmic ipolesis is a phenomenon closely associated with blood cells. Am J Vet Res 45: 769-770, 1984. hyperplasia of hematopoietic cells, especially, an 12. Shamoto, M: Emperipolesis of hematopoietic cells increase of megakaryocytes as host cells and in myelocytic leukemia. Virchow Arch B 35: 283 -290, 1981. neutrophils as entering cells in the bone marrow. 13. Richters, A, Sherwin, RP, and Richters, V: The lymphocyte and human lung cancers. Cancer Res Acknowledgement: The authors would like to thank Dr. 31: 214-222, 1971. Kunio Doi (Department of Biomedical Science, Faculty of 14. Sobolewski, S: Phagocytosis by megakaryocytes in Agriculture, The University of Tokyo) for reviewing the malignant disorders. Br J Haematol 45: 173, 1980. manuscript. 15. Parmley, RT, Kim, TH, Austin, RL, Avarado, CS, and Ragab, AH: Emperipolesis of neutrophils by dysmorphic megakaryocytes. Am J Hematol 13: 303-311, 1982. References 16. Takaya, M and Takahashi, K: Emperiolesis in a 1. Humble, JG, Jayne, WHW, and Pulvertaft, RJV: case of malignant lymphoma: electron microscopic Biological interaction between lymphocytes and and immunohistochemical investigation. Ultras other cells. Br J Haematol 2: 283-294, 1956. truct Pathol 12: 651-658, 1988. 2. Tavassoli, M: Emperipolesis by megakaryocyte in 17. Rozman, C and Vives-Corrons, JL: On the alleged blood loss. Br J Heamatol 49: 660-661, 1981. diagnostic significance of megakaryocytic 3. Thiele, J, Krech, R, Choritz, H, and Georgii, A: phagocytosis (emperipolesis). Br J Haematol 48: Emperipolesis-peculiar feature of megakaryocytes as 510, 1981. evaluated in chronic myeloproliferative diseases by 18. Cashell, AW and Buss, DH: The frequency and morphometry and ultrastructure. Virchows Arch B significance of megakaryocytic emperipolesis in 46: 253-263, 1984. myeloproliferative and reactive states. Ann 4. Larsen, TE: Emperipolesis of granular leukocytes Hematol 64: 273-276, 1992. with in megakaryocytes in human hematopoietic 19. Tanaka, M, Aze Y, and Fujita, T: Unpublish deta. bone marrow. Am J Clin Pathol 53: 485-489, 20. Sahebekhitiari, HA and Tavassoli, M: Marrow cell 1970. uptake by megakaryocytes in routine bone marrow 5. Tavassoli, M: Modulation of megakaryocyte emper smears during blood loss. Scand J Haematol 16: ipolesis by phlebotomy: Megakaryocytes as a com 13-17, 1976. 21. Takagi, H, Shimoji, N, Ogasawara, H, Noguchi, O, ponent of marrow-blood barrier. Blood Cells 12: 205-216, 1986. Fujii, H, Murata, A and Harada, Y: Modification 6. Lee, KP: Emperipolesis of hematopoietic cells of pathologic changes in toxicological studies due to within megakaryocytes in bone marrow. Vet Path endotoxin derived from intestinal flora. J Toxicol ol 26: 473-478, 1989. Pathol 6: 89-104. 1993. 7. Chen, LT, Handler, ES, and Weiss, L: An electron 22. De Pasquale, A, Paterlini, P, Quaglino, D, and microscopic study of the bone marrow of the rat in Quaglino, D: Emperipolesis of gralulocytes within megakaryocytes. Br J Haematol 60: 384-386, 1985.