MONOCYTES FROM CIRCULATING BLOOD FUSE IN VITRO WITH PURIFIED OSTEOCLASTS IN PRIMARY CULTURE

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1 J. Cell Set. 66, (1984) 335 Printed in Great Britain The Company of Biologists Limited 1984 MONOCYTES FROM CIRCULATING BLOOD FUSE IN VITRO WITH PURIFIED OSTEOCLASTS IN PRIMARY CULTURE A. ZAMBONIN ZALLONE, A. TETI AND M. V. PRIMAVERA Institute of Human Anatomy, University of Bari, Policlinico, Ban, Italy SUMMARY The origin of osteoclasts from mononuclear phagocytes and the addition of new nuclei to already differentiated osteoclasts have already been documented by several authors, but the factors controlling these events have not yet been elucidated. With the aim of investigating this problem, monocytes, isolated from circulating blood of laying hens and cultured previously for 5 days, were added to osteoclasts isolated from the medullary bone of the same hen and cultured at low density. The cultures were either fixed and observed under phase contrast at 24-h intervals for 5 days or filmed by time-lapse cinemicrography. With both techniques the formation of extensive areas of membrane contacts, generally followed by fusion between some monocytes and osteoclasts, was observed. The absence of added resorbing factors and the possible mechanisms by which osteoclast precursors are recruited in vivo are discussed. INTRODUCTION The origin and fate of osteoclasts are among the most puzzling problems of bone biology. The concept that they may be derived from a monocyte or macrophage precursor was proposed many years ago. Hancox (1949), in a review of the earlier literature, noticed many characteristics common to mononuclear phagocytes and osteoclasts, i.e. motility, undulating membranes and affinity for the supravital dye neutral red. Young (1962) showed, in experiments performed using tritiated thymidine, that they do not form by mitotic division but by fusion of mononuclear cells. Experimental evidence that they derive from mononuclear leukocytes was provided first by Fishmann & Hay (1962) and then by Jee & Nolan (1963), but it was not until several years later that further studies were undertaken in this direction. Buring (1975) and Gothlin & Ericsson (1976), using grafting techniques, provided further evidence of the blood-born nature of the osteoclast precursors. The same results, using quail-chick chimerae, were obtained by Kahn & Simmons (1975) and Jotereau & Le Douarin (1978). Several studies performed on osteopetrotic animals showed that osteoclast precursors could be found in blood (Walker, 1972, 1973; Marks, 1976; Toyama, Moutier & Le Mendin, 1974), bone marrow (Walker, 1975; Loutit & Sansom, 1976) and thymus or spleen (Walker, 1975; Marks & Schneider, 1978). Convincing evidence that osteoclasts are formed by fusion of mononuclear blood-carried cells, namely monocytes, was furnished by Tinkler, Linder, Williams & Johnson (1981). These authors inoculated intravenously tritiated-thymidinelabelled blood monocytes, harvested from the blood of donor mice, into syngenic

2 336 A. Zambonin Zallone, A. Teti and M. V. Primavera recipient animals in which osteoclast formation was being stimulated by 1-a-hydroxycholecalciferol. They found labelled osteoclasts in autoradiographs prepared from the femurs of recipient mice, demonstrating that during hormonally stimulated osteoclast formation, blood monocytes form one source of osteoclasts. The aim of the study reported here was to observe directly, using in vitro cultures, whether monocytes can fuse with pre-existing osteoclasts and, if so, the conditions under which this event can occur, i.e. if some kind of stimulation is necessary or if it will occur spontaneously. A preliminary report and the time-lapse cine-films were presented at the XVII European Symposium on Calcified Tissues (Zambonin Zallone, Teti & Primavera, 1983). MATERIALS AND METHODS Osteoclast cultures Osteoclasts were isolated from medullary bone of laying hens as described elsewhere (Zambonin Zallone, Teti & Primavera, 1982). With a view to the later addition of monocytes, osteoclasts were plated at low density (0-8 X K^/lOOmm dish) onto plastic tissue-culture dishes with or without glass coverslips. Osteoclasts were kept in MEM + 10% foetal calf serum and incubated at 37 C in a water-saturated atmosphere of 95 % air and 5 % CO2. Five days were allowed for complete spreading before adding the monocytes. Monocyte cultures Monocytes were prepared from freshly drawn hepannized blood of laying hens by Ficoll- Hypaque sedimentation (density g/ml) according to a published technique (Bennet & Cohn, 1966; Drew et al. 1978). Cells were plated at a density of 10 5 per 35 mm dish under the same conditions used for the osteoclasts. After 5 days of culture monocytes were detached from the dish with a solution of trypsin(0-25 %)/EDTA(0-1 %) /PBS (1: 1: 2, by vol.) and added to the osteoclast cultures at a concentration of 5 X 10 5 per 100 mm dish. At 24-h intervals, coverslips with cells were fixed for 5 min at room temperature in 3-7 % formaldehyde, directly mounted in 90 % phosphatebuffered glycerol and observed in a Zeiss Universal microscope equipped for phase contrast, while cells cultured in flasks without coverslips were used for time-lapse microcinematographic observations. After 5 days of co-culture all the cells were fixed. Time-lapse cinematographs were taken using a Crouzet variotimer with a Leitz inverted microscope and a Leicina special super 8 mm movie camera (Leitz) in a 37 C room with a 20-s interval between groups of six exposures. A suitable field was chosen and filmed on Kodak Ektachrome E 160 through a 32 X phase-contrast objective continuously for 7h. RESULTS Phase-contrast observations After 5 days in vitro osteoclasts cultured at low density formed a discontinuous layer of disk-shaped cells flattened on the substrate. Monocytes added to these cultures were at the beginning randomly distributed but after a few hours many of them were seen preferentially encircling large osteoclasts (Fig. 1). Some monocytes appeared round and flattened on the substrate while others exhibited irregular shape and an undulating membrane at one side or at the cell poles. Although rarely observed after 24 h, various kinds of contacts between monocytes and osteoclasts became very frequent in the following days, suggesting that fusion was

3 Blood monocytes fuse in vitro with osteoclasts 337 \ t \ \ Fig. 1. Phase-contrast microphotograph of 24-h-old joint culture of osteoclasts and monocytes. A large flattened osteoelast (oc) is surrounded by several monocytes displaying undulating membranes (arrows). X200. occurring between the cells involved. Some monocytes showed filopodia contacting neighbouring osteoclasts and/or cytoplasmic processes were pushed out by osteoclasts toward the monocytes (Fig. 2A, B). In other cases the plasma membranes of the two cells appeared closely apposed over a large area (Fig. 2c, D) or, possibly, in a later stage, the membranes seemingly disappeared from the place of contact and the monocyte formed a kind of protrusion on the osteoelast surface (Fig. 2E, F). Furthermore, in the outer cytoplasm of some osteoclasts, single nuclei surrounded by Fig. 2. Four-day-old joint culture of osteoclasts (oc) and monocytes. Phase-contrast microphotographs of a reconstructed sequence of events during the process of fusion. A-D. In an early phase contacts of various extension between the surface of the two cells can be observed, E-H. After the fusion of the two membranes the nucleus and the surrounding cytoplasm of the monocyte move progressively toward the osteoelast centre. X672. Fig. 3. Two-day-old joint culture of osteoclasts and monocytes. Microphotographs reproduced from time-lapse microcinematography. The squares localize a monocyte in fusion with the osteoelast. A, B. Early phase of contacts, C-E. The monocyte appears progressively smaller probably because the fusion is in act on the basal surface of the cell. F. The area of fusion is still recognizable because higher of the surrounding cytoplasm. G, H. The fusion is complete, the cytoplasm in the area of fusion keeps moving actively: confront the different shape of the cell edge. Time intervals are shown (in min). X160.

4 33 A. Zambonin Zallone, A. Teti and M. V. Primavera 2A OC Fig. 2. For legend see p. 337.

5 Blood monocytes fuse in vitro with osteoclasts Fig. 3. For legend see p. 337.

6 340 A. Zambonin Zallone, A. Teti and M. V. Primavera an array of dense granules were visible (Fig. 2G, H), suggesting recent fusion, but not yet mixing, of the cytoplasm of the monocyte and the osteoclast involved. Time-lapse microcinematography Time-lapse microcinematography confirmed and explained the observations on fixed cultures. Monocytes were seen to be actively mobile around osteoclasts. Continuous formation and retraction of lamellipodia and filopodia were observed. The cytoplasm of the osteoclast facing the monocyte was also in active movement. In some cases (3 monocytes out of 30 present in the field) after 2 3 h of making contact and detaching the monocyte fused with the cytoplasm of the osteoclast (Fig. 3). The part of the cytoplasm of the osteoclast where the fusion with the monocyte took place kept moving considerably for another hour. In other cases (6 monocytes out of 30 present in the field) after the first phase of contacts the monocyte moved away from the osteoclast without cell fusion occurring. DISCUSSION The recently developed technique for isolating a fairly pure population of osteoclasts has now allowed the observation of these cells in vitro (Zambonin Zallone, Teti & Primavera, 1982, 1983; Zambonin Zallone et al. 1983) and provides a tool for gaining insight into the biology of osteoclasts. We have been able to visualize in this way the process of fusion of monocytes with osteoclasts that have already differentiated. We believe that we have observed specific fusion and not phagocytosis, because phagosomes containing nuclei were never observed. Furthermore, other cell types (such as lymphocytes or neutrophils) occasionally present in the culture were never seen in the process of fusion with osteoclasts. Jaworski, Duck & Sekaly (1981), using tritiated thymidine and sequential biopsies, demonstrated that the average life of a single nucleus in the osteoclast is 9-11 days and that the osteoclast nuclei are continuously renewed. However, we do not know what determines either the number of nuclei present in an osteoclast or their renewal; in our population the number of nuclei contained in an osteoclast ranges between 2 and 30, with the highest frequencies in the range from 10 to 20. This number could very well depend upon the number of available monocytes. In experiments in vitro, resorbing matrix has been demonstrated to be chemotactic for monocytes (Mundy et al. 1978; Maloneeia/. 1982; Mundy & Poser, 1983); in vivo, the presence of bone being resorbed could induce the migration of monocytes from blood to the place of active resorption. We do not know whether all circulating monocytes can differentiate into osteoclasts. Certainly in our cultures in the absence of added resorbing factors, only some monocytes enter the osteoclasts, while others, after a preliminary period of membrane contacts, move away. Monocytes that appear identical at the lightmicroscopic level have already been shown to have different characteristics and functional capabilities (Roubin & Zolla-Pazner, 1979). In vivo factors that systemically induce bone resorption and increase the number of osteoclasts could determine the

7 Blood monocytes fuse in vitro with osteoclasts 341 liberation from the marrow into the blood of cells apt to fuse with osteoclasts, while diffusible products of bone resorption could act locally indicating to circulating monocytes the sites where resorption is taking place; however, further work is necessary to substantiate this attractive hypothesis. This work was supported by C.N.R. and M.P.I, research grants to A.Z.Z. REFERENCES BENNET, W. E. & COHN, Z. A. (1966). The isolation and selected properties of blood monocytes. 7. exp. Med. 123, BURING, K. (1975). On the origin of cells in heterotopic bone formation. Clin. Orthop. &Rel. Res. 110, DREW, S. I., NATHANSOND, S., ZAMFIRESCU, P., CARTER, B. M., TERASAKI, P. I., NAIEM, F. & BILLING, R. (1978). Serological characterization of human monocytes for HLA, B-lymphocytes, granulocytes and monocyte-associated antigens by cytotoxicity testing. Tissue Antigens 11, FISHMANN, D. A. & HAY, E. D. (1962). Origin of osteoclasts from mononuclear leukocytes in regenerating newt limbs. Anal. Rec. 143, GOTHLIN, G. & ERICSSON, J. L. E. (1976). The osteoclast. Review of ultrastructure, origin and structure-function relationship. Clin Orthop. &Rei. Res. 120, HANCOX, N. M. (1949). The osteoclast. Biol. Rev. 24, JAWORSKI, Z. F. G., DUCK, B. & SEKALY, G. (1981). Kinetics of osteoclasts and their nuclei in evolving secondary Haversian system. J. Anal. 133, JEE, W. S. S. & NOLAN, P. D. (1963). Origin of osteoclasts from the fusion of phagocytes. Nature, Lund. 200, JOTEREAU, F. V. & LE DOUARIN, N. M. (1978). The developmental relationship between osteocyte and osteoclasts: a study using Quail-chick nuclear marker in endochondral ossification. DeviBtol. 63, KAHN, A. J. & SIMMONS, D. J. (1975). Investigation of cell lineage in bone using a chimaera of chick and quail embryonic tissue. Nature, Land. 258, LOUTIT, J. F. & SANSOM, J. M. (1976). Osteopetrosis in microphthalmic mice - a defect of the haematopoietic stem cell? Calc. Tiss. Res. 20, MALONE, J. D., TEITELBAUM, S. L., GRIFFIN, G. L., SENION, R. M. & KAHN, A. J. (1982). Recruitment of osteoclast precursors by purified bone matrix constituent. J. Cell Biol. 92, MARKS, S. C. JR (1976). Tooth eruption and bone resorption. Experimental investigation of the ia (osteopetrotic) rat as a model for studying their relationships. J. oral Path. 5, MARKS, S. C. JR & SCHNEIDER, G. B. (1978). Evidence for a relationship between lymphoid cells and osteoclasts: bone resorption restored in ia (osteopetrotic) rats by lymphocytes, monocytes and macrophages from a normal littermate. Am.jf. Anat. 152, MUNDY, G. R. & POSER, J. W. (1983). Chemotactic activity of the y-carboxyglutamic acid containing protein in bone. Calcif. Tiss. Int. 35, MUNDY, G. R., VARANI, J., ORR, W., GONDEK, M. D. & WARD, P. A. (1978). Resorbing bone is chemotactic for monocytes. Nature, Land. 275, ROUBIN, R. & ZOLLA-PAZNER, S. (1979). Markers of macrophage heterogeneity. I. Studies of macrophages from various organs of normal mice. Eur.J. Intmun. 9, TINKLER, S. M. B., LINDER, J. E., WILLIAMS, D. M. & JOHNSON, N. W. (1981). Formation of osteoclasts from blood monocytes during 1 a-oh-vit. D stimulated bone resorption in mxct.j. Anat. 133, TOYAMA, K., MOUTIER, R. & LE MENDIN, H. (1974). Resorption osseuse apr6s parabiose chez les rats "op" (osteopetrose). C.r. hebd Seanc. Acad. Sci., Paris, D 278, WALKER, D. G. (1972). Enzymatic and electron microscopic analysis of isolated osteoclasts. Calc. Tiss. Res. 9, WALKER, D. G. (1973). Osteopetrosis cured by temporary parabiosis. Science, N.Y. 180, 875.

8 342 A. Zambonin Zallone, A. Teti and M. V. Primavera WALKER, D. G. (1975). Control of bone resorption by haematopoietic tissue. The induction and reversal of congenital osteopetrosis in mice through use of bone marrow and splenic transplant. J.exp.Med. 142, YOUNG, R. W. (1962). Cell proliferation and specialization during endochondral osteogenesis in young rats. J. Cell Biol. 14, ZAMBONIN ZALLONE, A., TETI, A. & PRIMAVERA, M. V. (1982). Isolated osteoclasts in primary culture: first observations on structure and survival in culture media. Anat. Embryol. 165, ZAMBONIN ZALLONE, A., TETI, A. & PRIMAVERA, M. V. (1983). Spontaneously occurring fusion of monocytes from circulating blood with osteoclasts cultured in vitm. Calcif. Tiss. hit. (suppl.) 35, A20, 74. ZAMBONIN ZALLONE, A., TETI, A., PRIMAVERA, M. V., NALDINI, L. & MARCHISIO, P. C. (1983). Osteoclasts and monocytes have similar cytoskeletal structures and adhesion property in vitro. J. Anat. 137, (Received J August J983-Accepted 30 September 1983)