Phagokinetic properties of human mononuclear phagocyte cell cultures

Experimental Cell Research 133 (1981) 127-13

INETIC PROPERTKES OF PHAGOCYTE CELL KATHERINE

M. KOWALCHYK,

Department

of Microbiology

STEVEN H. ZUCKERMAN’

and STEVEN D. DOLJCLAS’ 3

and Medicine, University o, Minnesotu I Mnneapolr~ L1 MN 55455, US:

Medico1 School,

SUMMARY Random movernen? of human peripheral blood monocyte and pulmonary alveolar macrophage (PAM) cultures was examined using the phagokinetic track assay. The track type of the two cell types was distinct; PAMs cleared larger areas of the colloidal gold. Both cell populations exhiSited increases in phagokinesis by 1 week of in vitro culture; PAM cultures by 1 day of in vitro culture. Alteration of the substratum by coating with BSA-anti BSA immune compiexes resulted in the inhibition of the movement of monocytes but not PA%. Electronmicroscopy and microcinematography were used to study cell-substratum interactions.

Phagokinesis defines a process of cell loco- bovine capillary e~dothelial cells has been motion on colloidal gold-coated surfaces reported to be in ibited by i~te~e~on R ich results from phagocytosis and random migration [l]. Albrecht-Buehler developed the technique to study particle macrophage cell movement in the plasma membrane, and based primarily o has since used the method to evaluate cell Macrophages activated with locomotion and spreading [2]. Studies with 3T3 cells have demonstrated that particle pinocytosis, chemo removal from the substratum accompanies acrophage movement ement and is accomplished by filoa, lobopodia and lamellopodia [3-61. As the gold is cleared from the substratum, a o&ion becomes internalized while the majority accumulates n the dorsal surface of the cell. The cleare track which results by colchicine, whereas colchicine plus is a record of the cell’s movement and can cytochalasin I3 inhibits total cell movement be used to uantitate cell movement. as evaluated by migration from capillary piPhagokinetic tracks of SV40 and polyomatransformed 3T3 cells, a hamster cell line 1 Present address: Department of Medical Ceil Gent-rits, Karolinska Enstitutet S-10401 Stockholm 60. NIL-II, sarcoma virus-transformed NIL-8 Sweden. cells and human neutrophils have been re- * Division of Allergy-Immunology, Children's HOS34th Str. and Civic Center Bd, Philadelphia, ported [I, 7, 8, IO]. Recently, using the p&al, PA191043USA. ~~agoki~et~c track assay, the movement of 3 To whom offprint request should be addressed. 9-811813

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pettes and by microcinematography [ 13161. Microcinematography has also been used to develop mathematical models of human granulocyte movement [17, 181. These assay systems are limited since they do not allow for analysis of a large number of individual cells in the population, as currently available with the phagokinetic track assay. Phagokinetic measurements of human monocyte-macrophage cell populations have not been reported. We have recently described a system which permits the long-term in vitro culture of human peripheral blood monocytemacrophages [21]. Therefore, the present studies were designed to determine whether significant changes in random cell movement occur during in vitro growth and differentiation of monocytc-macrophage cell populations. In addition, we have assessed qualitative and quantitative changes in phagokinesis in these two cell types. We report that both cell types exhibit phagokinesis and that pha.gokinesis increases during in vitro culture. Furthermore, whereas membrane colloidal gold interactions in both systems were mediated by similar pseudopodal-like processes, human monocyte but not pulmonary macrophage phagokinesis was reduced on immune complex-coated coverslips. MATEKIALS

AND METHODS

Colloidal gold precipitate was prepared as described by Albrecht-Buehler [I]. Briefly, I.8 ml of 14.5 mM AuCI,H (Baker), 6 ml of 36.5 mM Na,C03 (Baker) and 1 I ml of H,O were mixed and brought to a boil. Immediately upon boiling, I.8 ml of 0.5% formaldehyde (L;SP. 33%) was added. resulting in a brown preclpitate. Circular cover5lips (Bellco, IS mm 0) were prepared by dipping into I ‘/c bovine serum albumin (BSA) (Sigma), followed by 100%~ ethanol and then dried with a hot air dryer. Hot colloidal gold precipitate, I .5 ml per coverslip was added to 16 mm Linbro wells and incubated for 1 h in S% CO. incubator at 37°C. Immune complex-coated coverslips were prepared by incubating BSA-coated covcrslips with a l/25 dilu-

Erp C‘rll t3c.s 1.H (/W/J

in phosphate-buf1’eon of 7S anti-BS.4 (Miles-Ycda) fercd saline (PBS) for I h at 22°C 1191. Coverslips were then washed with PBS, and coated with colloidal gold. Colloidal gold-coated coverslips were washed three times, prior to cell addition. with Dulb~cco’s Modified Eagle ,Mcdium (DEIM) with 10% fetal calf serum (KS) and 10% horse serum (HS).

Cell cu1tt~rc.s ctnd c.xp-l,erimental

design

Human peripheral blood monocytes were isolated. after Ficoll-Hypaque separation, hy adherence and clution from microcxudatc-coated tissue culture flasks [ 20. 211. Routinely. 10-16x 10’ cells were obtained. 95% positive for latex ingestion and non-specific estcrasc stain. Human pulmonary alveolar macrophagcs (PAMs) were collected from smokers by fiber optic bronchoscopy in physiological saline. Both cell types were rcsuspcnded in DEM supplemented with 10% FCS, 10% HS. 4 mm glutamine, penicillin (100 units/ml), streptomycin (IO0 &ml) and non-essential amino acids. Cells were plated at 2X IV cells per I6 mm Linbro well and maintained at 37°C in 5 % COz and 100% humidity. Monocyte-macrophage cultures can be maintained up to 4 months with approx. 50% of the cells recoverable at any time point as non-adherent cells by washing adherent cultures 1211. Non-adherent cells exhibited a typical monocyte-macrophage morphology, retained Fc receptors. ingested latex, and when placed in fresh Linhro wells became adherent. Approx. 95 % of the non-adherent cells excluded trypan blue. Ken-adherent cells from I 11) 21 day cultures were removed and IO’ cells incubated on colloidal gold-coated coverslips at 37’C. Random fields were photographed at I, 7-8. and 20 h with a Zeiss inverted phase-contrast photomicroscope. Track lengths were measured with a map measure and track areas h! planimctry. ‘fracks without a cell associated were not measured. All data arc expressed in pm, each time point is representative of at least three experiments with cells from different donors: at least 20 cells were evaluated at each time point.

Scanning and transtnissiot~ electron microscopy (SEA4 rrnd 7’EM) After l-3 h of incubation. coverslips wcrc washed in DEIM. followed by fixation in I .S% glutaraldehyde in 0. I M sodium cacodylatc with 6% sucrose at 22°C for IS min. Samples were then serially dehydrated. critical point-dried, and examined using a Hitachi Scanning Electron Microscope. Rcprescntativc photomicrographs were taken at a magnification of x%&IS(?Go. For TEM, coverslips were fixed in 1.5% glutaraldehydc in sodium cacodylate at 4°C overnight, followed by treatment with I c/ 0~0, in 0.133 N sodium cacodylate for 20 min. Specimens were stained with I ‘X uranyl acetate for 20 min and processed for elecFig. I. Phagokinetic tracks of monocyte-macrophages. after 20 h on colloidal gold. ((I) Freshly, isolated monocytes; (6) monocytcs maintained in vitro for 7 days; (c) freshly isolated PAlvls; ((/) PAMs maintained in vitro for 7 days. X450.

hagokinetic properties of human ~o~~~~~~e~~phagocyte celEcdtures

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Fig. 2. Histogram of phagokinetic track lengths (a, c) and phagokinetic areas (b, d). In each group, measurements were determined for at least 20 cells in 3 experiments with different donors. (a, b) Monocytes;

(c, d) PAM?.; (A) Freshly isolated cells; (B) 7-day cells; (C) I4day PAMs or day 21 monocytes. Arrows indicate median track length or area.

Phagokinetic properties of human mononuclear phagocyte cell cultus-es

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show increases in track lengths, with track pm. A similar trend was observed when area measurements were coimpared. y day 7, 25% of the cells have cleared areas >I80 pm2, wit increased Z-fold (fig. 26). At day 214 significant differences ‘were seen w

Fig. 3. Cells with track lengths >50 pm, O-O, Peripheral blood monocyte; O--O, PAM; n=3.

ve increased trac >420 prn”~ Similar results were obtai PAM cultures (fig. 2~). After 7 days, PAMs demonstrate no increase in total track a 2-fold increase y day 14, 35% of

tron microscopy (221. Sections were stained with lead citrate and photographed at final magnifications of x3000-:5000.

Time-lapse photography Randomlv chosen cells were observed for uo to 7-8 h by time:lapse photomicroscopy. Covershps were olaced in Svkes-Moore Chambers in 1 ml of medium. and maintained at 37°C. Photographs were taken, i frame/IO set on Kodak Tri-X Reversal 16 mm film.

Human monocytes and pulmonary alveolar macrophages were capable of phagokinesis (fig. I). Increase in total track length and areas were observed in both cell types following in vitro culture for 7 days. PAMs, however, consistently cleared larger areas of the colloidal gold. A~t~o~g~ the cell populations contained >90 % r~o~o~~ciear phagocytes, quantitative measurements of track lengths and areas demonstrated heterogeneity in cell movement (fig. 2). Pn general, 20-30% of total cell population demonstrated nges in phagokinetic properties during in vitro culture. By day 7 of monocyte culture, 30% of the cells have increased track lengths greater than 100 pm and median is increased Z&fold (fig. 2a). At 21 days, 20 % of these cells continued to

onstrate a 3-fold increase in median area n contrast to the monocyhes, by ave increased mcrease m median are2, Cells of both ined CJuman rno~~cy~es did not show increase in track lengths untii day 6. whereas a substantial PA s~~~~~~~at~~~ had increased track lengths after 24 11. Finally, ~bag~ki~es~s of ~r~s~~y-~s~~ate~ monocytes was shown to be inhibited on immune complex-coate covers@3 (fig. 4): 35% of the cells had decreased track lengths) wit a 2-fold decrease iri track length. At y 7, less hnhibiti seen; 20% of cell creases in track le smission electron Scanning and and TEMJ microscopy (S SEM revealed that acted with the goldsimilar manner (fig. 5). The majority of gold particles were brought to the cell surface

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Fig. 4. Histogram of phagokinetic track lengths of monocytes incubated on BSA-anti BSA immune complexes. (a) Freshly isolated cells (control, fig. 2 aA); (b) 7-day cells (control, fig. 2 a@.

by membrane projections 0.2 pm in diameter and up to 50 pm in length. Occasionally, flat sheet-like membranous projections could be seen covering gold particles. Most of the gold appeared to remain on the cell surface and was located to the rear of the cell in a ‘cap’-like formation. TEMs also showed the ‘cap’-like accumulation of gold particles at the rear of the cell and confirmed that relatively little gold was internalized by either cell type (fig. 6). Time-lapse photography

A common feature among all cells observed was clearance of a peripheral circular area Exp CellRes

133 (19811

of gold within 10 min. The gold was mainly cleared by membranous projections which transported gold to the cell body by reverse flow that often appeared to involve retraction into the cell body. Once this peripheral circular area was cleared, the cells began to move. Movement was accompanied by the accumulation of gold particles on the cell surface and formation of a ‘cap’ at the rear of the cell. The leading edge remained relatively free of particles, and especially in the case of cells cultured in vitro for a week or more, appeared to extend pseudopodial projections towards the substratum while dragging along the rear cap of gold particles. No significant differences in the manner of movement were observed between the two cell types. However, there were differences seen when freshly-isolated cells of either type were compared with cells which had been cultured in vitro for a week or more. Freshly-isolated cells had fewer membranous projections and less gold was accumulated on the surface of the cells. Although velocities were not quantitated, day 0 cells appeared to move slower than cultured cells and showed movement for only 3-4 h, compared with >8 h for cultured cells. The freshly-isolated human monocytes were more sensitive to gold toxicity as once they stopped moving, most

Fig. 5. (a) SEM of 7-day monocyte on colloidal gold for 2 h; note prominent membrane projections (UP rows) with attached particles. (b) Cell incubated as in (a) showing lobopodia; (c) higher magnification of cell in (a) showing microspike-gold interaction; (d) higher magnification of cell in (b) showing interaction with colloidal gold. In most instances, gold appears to localize at rear of cell body. (a) x1750; (b) x915; (c) x15500;(d) x6000. Fig. 6. (u) TEM of 7-day monocyte. Colloidal gold particles are seen close to plasma membrane, rarely internalized. (6) Higher magnification of (a) showing interaction between plasma membrane and colloidal gold particles. (a) x4375; (b) x25000.

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cells detached or lysed within 30 min. This was not seen with cells cultured in vitro.

track lengths and areas for longer time periods. The time-lapse photographic observations are in some respects consistent with this interpretation. Both freshly-isoDISCUSSION lated monocytes and PAMs move for much Human PAMs and monocytes show in- shorter time periods than cultured cells and creases in phagokinesis during in vitro cul- many cells lyse 30 min after the cells stop ture. A possible explanation for this obser- moving. Toxicity was a limitation in this vation may be that the cells are ‘stimulated system as none of the cell types were able in vitro by factors in the media. Serum is to continue moving past 20 h; however, up required for maximal movement of mono- until that time, the cells were actively nuclear phagocytes and 3T3 cells [3]. Alter- motile despite the fact that by 3-4 h the cells natively, increased phagokinesis may re- have accumulated large amounts of gold. flect in vitro macrophage differentiation. Time-lapse photography and EM studies Monocytes by 3-5 days culture undergo of both cell types showed that similar to changes which include increases in leucine previous studies of fibroblasts [4, 51, the and uridine uptake, changes in cell size and majority of the gold was removed via micromorphology, increases in acid phosphatase spikes in a reverse flow manner and acand adenosine kinase activity, and changes cumulated at the rear of the cells in a capin adenosine deaminase activity [21,23,24]. like formation, while the leading edge reIn the present study, monocytes did not mained free of particles. However, there is show increases in phagokinesis until after no evidence of active recycling of the memthese changes at day 6, whereas PAMs brane as the TEMs show that little of the show increases in phagokinesis by 24 h cul- gold is internalized, and few membrane vesture. The increase in track lengths and icles are seen. Whether membrane at the areas by PAMs may reflect in vivo or in leading edge is newly synthesized or revitro ‘activation’, as these cells were ob- cycled cannot be resolved. Our observatained from smokers [25,26]. The increased tions are consistent with the model for cell area cleared by PAMs may relate to an in- locomotion suggested by Rajarman et al. crease in their phagocytic capacity com- that describes cell locomotion as a unique pared with monocytes, as track lengths of form of capping where the ceil repeatedly both cell types were similar. Others have discards the substratum [29]. When the cells demonstrated increases in the phagocytic successfully adhere to the substratum, such ability of ‘activated’ macrophages [27, 281. as glass or plastic surface, the cell would Our electron microscope studies, however, move away from the substratum by ‘capdo not support this conclusion as little of ping’ the adhesion sites toward the posthe gold is internalized by these cells. An terior end, resulting in the displacement of alternative interpretation is that the en- the cell body in the opposite direction. hanced phagokinesis seen during prolonged This model predicts the appearance of gold in vitro culture is not due to increased ‘cap’ at the rear of the cell, while the leadability to move, but to a decrease in the ing edge remains free of particles. The effect of a different substratum on cell’s susceptibility to gold toxicity. If so, the cells may become less sensitive to gold phagokinesis was examined to further intoxicity and then can continue to increase vestigate the role of substrate-membrane Exp Cell Rcs 133 (1981)

hagokinetic

properties

of human

interactions and the subsequent effects on cell locomotion. Human monocytes plated on immune complex gold-coated coverslips exhibited partial inhibition of phagokinesis. Fc receptor antibody interaction may lead to increased cellular adhesiveness to the substratum by receptor-ligand cross-linking as suggested by studies of ConA and other ) 3 11. It has been reported that increases in cell adhesion to the substratum inhibit migration [32, 331. Alternatively, Fc receptor-ligand interaction may trigger an event leading to a change in the micr~fi~arne~~ organization such that comotion is inhibited. Since neither y-isolated nor cultured PAMs are inhibited on immune complex-coated coverslips, it is not yet known whether the difetween monocytes and PAMs relate to receptor-ligand interaction or to metabolic d~~ere~ces. A s~r~~isi~g findi was that the capacity of cells to move in s system was heterous, even though both monocytes and cell populations were greater than 90 % pure as assessedby latex ingestion and peroxidase staining. This heterogeneity, however, was also observed in freshlyisolated cells, an therefore it appears unlikely that it is d e primarily to the use of the ~~~-ad~er~~~ cell population. To further assess this possibility, lidocaine was used to remove adherent cultured cells. Cells removed in this manner demonstrated similar heterogeneity in track lengths and areas comparable to non-adherent cells (data not shown). In addition, it is important to note that the non-adherent cells constitute approx. 50% of the total cell ~~p~Iat~o~~ ingest latex, are peroxidase positive and become adherent when placed in fresh Linbro wells. Thus, it is possible at the heterogeneity is related to the existence of mononuclear phagocyte sub-

rnononuclenr

phagocyte

cell cultures

135

populations [34] and not necessarily due to in vitro selection. Despite possible difficulties wit measurements of phago inesis are valuable in assessi serve as a me can be used to further inves anism of cell mo

large number of individual cells within a population, will contribute to the study of Cdl

~QCQITKdiQn.

These studies were suooorted by USPHS m-ant KIII the Kroc Foundation, and the National Foundation-March of Dimes (6-244). S. H. Z. is a Postdoctoral Fellow of the American Lung Assoc:ation. K. %I. K. is a Predoctoral student supported by NCI Training Grant 1T32CAO9138. We would like to thank Dr Murray Rosenberg for use of time-lapse photography equipment.

AI-12478,

1. Albrecht-Buehler, G, Cell 11 (1977) 395 2. Albrecht-Buehler, 6 & Yarneh, M M. Exp cell res 78 (1973) 59. 3. Albrecht-Buehler, C & Lancaster, R M, ? celi bioi 71 (1976) 370. 4. Albrecht-Buehier, @. I cell biol69 (1976) 275. 5. Albrecht-Buehler, G & Go?dman, R D, Exp ceil res 97 (1976) 329. 6. Albrecht-Buehler, 6, J ceil bioI72 (1977) 595. 7. Ah, I U & Hynes, R 0, Cell 14 (1978) 439. 8. Turner, S R, FASEB abstr (1978) 1169, no. 4974. 9. Zetter, B R, Nature 285 (1980) 41.

10. Brouty-Boye. D & Zetter, B R, Science 208 (1980) 516.

11. Poplack, D G, Shern, N A, Chaparas. S D & Blaese, R M, Cancer res 36 (1976) 1233. 12. Wilkinson, P C, Immunobiology of the macrophage (ed D S Nelson) p. 349. Academic Press. New York (1976). 13. Bhisey, A N & Freed, 3 .I, Exp cell res 64 (1971) 419. 14. - Ibid 95 (1975) 376.

15. Cheung, II T, Cantarow, W D & Sundaradas, 6, Exp cellres 111 (1978) 95. 16. Allan, R B & Wilkinson, P C, Exp ceil res 1 ii (1978) 191. 17. Peterson, S C & Noble, P B, Biophys j ‘2 (1972) 1048.

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18. Hall, R L & Peterson, S C, Biophys j 25 (1979) 365. 19. Douglas, S D, Cell immunol 21 (1976) 344. 20. Ackerman, S K & Douglas, S D, .I immunol 120 (1978) 1372. ?l. Zuckerman, S H, Ackerman, SK & Douglas, S D, Immunology 38 (1979) 401. 22. Branson, S H, Exp cell res 65 (1971) 253. 23. Zuckerman, S H, Olson, .I M &Douglas, SD, Exp cell res 129 (1980) 281. 24. Zuckerman, S H & Douglas, S D, Cell immunol 56 (1980) 478. 25. Holt, P G & Heast, D, Proc sot exp med 142 (1973) 1243. 26. Harris, J 0, Swenson, E W &Johnson, J E, J clin invest 49 (1970) 2086. 21. Hamburg, S I, Manejias, R E & Rabinovitch, M, J exp med 147 (1978) 593.

Exp Cell Res 133 (1981)

28. Rhodes, J, J immunol 114 (1975) 976. 29. Rajarman, R, MacSween, J M & Fox, R A, J theor bio174 (1978) 177. 30. Sato, C & Takasawa-Nishizawa, K, Exp cell res 89 (1974) 121. 31. Gail, M H & Boone, C W, Exp cell res 70 (1972) 33. 32. Johnson, G S & Pastan, I, Nature new biol 236 (1972) 247. 33. Grinnell, F, J cell biol58 (1973) 602. 34. Arenson, E B Jr, Epstein, M B & Seeger, R C, J clin invest 65 (1980) 613.

Received August 13, 1980 Revised version received November 27, 1980 Accepted November 28, 1980

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