Differentiation of from normal human in liquid culture. Electron microscopy and cytochemistry.

D R Bainton, D W Golde

J Clin Invest. 1978;61(6):1555-1569. https://doi.org/10.1172/JCI109076.

Research Article

To study the various stages of human mononuclear maturation, we cultivated bone marrow in an in vitro diffusion chamber with the cells growing in suspension and upon a dialysis membrane. At 2, 7, and 14 days, the cultured cells were examined by electron microscopy and cytochemical techniques for peroxidase and for more limited analysis of acid phosphatase and arylsulfatase. Peroxidase was being synthesized in of 2- and 7-day cultures, as evidenced by reaction product in the rough-surfaced endoplasmic reticulum, Golgi complex, and storage granules. Peroxidase synthesis had ceased in and the enzyme appeared only in some granules. By 7 days, large macrophages predominated, containing numerous peroxidase-positive storage granules, and heterophagy of dying cells was evident. By 14 days, the most prevalent cell type was the large peroxidase-negative . Thus, peroxidase is present in high concentrations in immature cells but absent at later stages, presumably a result of degranulation of peroxidase-positive storage granules. Clusters of peroxidase-negative macrophages with indistinct borders (epithelioid cells), as well as obvious multinucleated giant cells, were noted. Frequently, the interdigitating plasma membranes of neighboring macrophages showed a modification resembling a septate junction--to our knowledge, representing the first documentation of this specialized cell contact between normal macrophages. We suggest that such junctions may serve as zones of adhesion between epithelioid cells.

Find the latest version: https://jci.me/109076/pdf Differentiation of Macrophages from Normal Human Bone Marrow in Liquid Culture

ELECTRON MICROSCOPY AND CYTOCHEMISTRY

DOROTHY F. BAINTON, Department of Pathology, University of California at Sant Francisco, California 94143 DAVID W. GOLDE, Department of Medicine, University of California at Los Angeles School of Medicine, Los Angeles, California 90024

A B S TR A C T To study the various stages of human INTRODUCTION mononuclear phagocyte maturation, we cultivated bone marrow in an in vitro diffusion chamber with Recent studies have convincingly demonstrated that the cells growing in suspension and upon a dialysis macrophages of inflammatory originate from membrane. At 2, 7, and 14 days, the cultured cells precursor cells in the bone marrow, are transported were examined by electron microscopy and cyto- as monocytes via the peripheral circulation, chemical techni(lues for peroxidase and for more and differentiate further in tissues and cavities to be- limited analysis of acid phosphatase and arylsulfatase. come large macrophages (1-3), epithelioid cells, and Peroxidase was being synthesized in promonocytes of multinucleate giant cells (4-7). Seeking to identify 2- and 7-day cultures, as evidenced by reaction pro- the precursor cells of mononuclear in duct in the rough-surfaced endoplasmic reticulum, human bone marrow, Nichols et al. (8) and Nichols Golgi complex, and storage granules. Peroxidase syn- and Bainton (9, 10) found that electron microscopy thesis had ceased in monocytes and the enzyme ap- and peroxidase cytochemistry afford several advan- peared only in some granules. By 7 days, large tages over light microscopy and routine electron macrophages predominated, containing numerous microscopy in the definitive identification of the peroxidase-positive storage granules, and heterophagy various developmental stages of immature leukocytes. of dying cells was evident. By 14 days, the most In addition, the stages of maturation could prevalent cell type was the large peroxidase-negative be more explicitly recognized. macrophage. Thus, peroxidase is present in high In normal human marrow, peroxidase is synthesized concentrations in immature cells but absent at later early, during the stage, and is localized stages, presumably a result of degranulation of peroxi- in all granules and organelles of the secretory ap- dase-positive storage granules. Clusters of peroxidase- paratus-i.e., cisternae of the rough endoplasmic negative macrophages with indistinct borders (epi- reticulum (RER)l and Golgi complex. It has been thelioid cells), as well as obvious multinucleated determined that the peroxidase-positive granules also giant cells, were noted. Frequently, the interdigitating contain the enzymes arylsulfatase and acid phos- plasma membranes of neighboring macrophages phatase and hence are modified primary lysosomes. In showed a modification resembling a septate junction- the monocyte stage, peroxidase production ceases and to our knowledge, representing the first documentation is no longer visible in the RER or Golgi complex, of this specialized cell contact between normal macro- and a second population of granules is produced. phages. We suggest that such junctions may serve as Formation of these peroxidase-negative granules con- zones of adhesion between epithelioid cells. tinues while monocytes are being transported in the blood, but their content is unknown (9, 10).

Address reprint requests to Dr. Bainton. Received for publication 31 October 1977 and in revised I Abbreviation used in this paper: RER, rough endoplasmic form 10 February 1978. reticulum.

J. Clin. Invest. C The American Society for Clinical Investigation, Inc., 0021-9738/78/0601-1555 $1.00 1555 Relatively little research has been dlevote(l to the for 9() mi (Fig. lOB), followed by treatmlen(t with 2% (Nf14)2S cytochemiiical clcaracteristics of the huimiiani m11on1o- (17). Subsequent processing. Enzvymc' p)re'parations were( p)ost- nuiiclear phagoeyte systemii in vivo afteir the l)loocl fixed fOr 1 h at 40C( in 1%/(. OSo4 in acc'tate-\eronal (Win- monocvte stage. However, the ecultivationi of normiial throp Laboratories, Evanston, Ill.) bufter, pH 7.6, withl Or bone mnarrow cells in a li(qLli(l me(diumii in a Marlbrook in without staining en) bloc in uranvl acetate for 60 min, ad( vitro difutsioni chamber hlas recenitly leen achieved, then proccsse(l and( (cxaminedl as dlescril)d'l (8, 9). and provi(les a convenienit meanis for stu(lving the differentiationi of this cell line in short-terim cutiltuires RESULTS (11, 12). This system is advantageous l)ecause cells The number and( (lifferential of' l)one can be eciltivated in the absence of exogenouis stimiula- couinits norimial tory substances anid are easily retrieved for analysis marrow cells grown in a liquid me(diu have Al- rea(dy b)een (11). In at 2 clays of' finetioni, cytogenetics, anidl cytochemi stry. Usinig this reporte(d brief,' (and up to 4 or .5 clays), the predlominiianlt cel linie is the metho(d of' clultture, Golde and Cline (11) were alble to idenitif'y the various maitiuratioiail stages ofinoriiial neuitroplhilie granuhlovte; thereafter, the p)rop)ortion of humiainla milonloniutclear phagocytes by morphological anid maerophages progressively increases uintil 1b 7-14 tlhexy are prepond(lerant. Tle.se staii i cytochemilical procedures tusing light microscopy, anid (lays, macro)phages intensely for a-naphthvl butrwate esterase, but peroxi- also denionistrated that miiacrophages miatuirinig in dcase activity hlias been nioted to with miaitiura- vitro suirf'ace receptors for IgG anidl are capal)le (lecrcasc possess tion (12). of actively phagocytizinig microorganisms (11, 12). The puIrp1ose of' the presenit studly was to characterize Localization of l)eroxida.sc int 2-, 7-, anid 14-daiy these imimiatuire and milatuire hulitian milonioniulclear cunltu rcs phagocytes by electron microscopy combined with peroxiclase and lysosomal einzymile localizationi. The Promonociltes. The promonocytes (Fig. 1) caii be previousi investigationis by Nielols and Bainitoni (9, 10), idlentifie(d without (liffictilty at 2 and( 7 clays, but wvere using noriiail human bone marrow and 1)100(1, afford rarie 1b 14 dclays (Table I). These imimiaiitiure cells ap- a basis for comparing in vivo cellular (liff'erenitiation peare(l similar to thiose observed in freshly ol)btai ned with that o)bserved in vitro after, 2, 7, or 14 aclys niorimial hIiiiumani bone marrow (8-10), except thiait the of' culture. In this report, we describe the fine struc- pitsi)lia memiibranie finiger-like extenisionis were niot seen tiural and enzymatic alterations which ocecur (luiring the in vivo. Peroxidclase \as l)eing synthesized dutiring the in vitro maturation of promoniocytes to large mnacro- iniitial stages of milatuiration, sinice reactivity was phages andl giant cells. visible as a floccuilent density in alli cisternae of' the RER, all Golgi c'isterinae, and(i in both imimiaiitiure M ETHODS acI(i miaitiure cytoplasm ic grantiles (m-300 nini). louocltecs. The milonobcyte differed firom the piro- Materials. Bone marrow was obtained from tbep)osterior iliac crests of five healthy, adlult volunteers. monocyte in several aspects: (a) Thc' ntcleuis l)ec'ame The histochetnical reageints, Grade I 8-glycerophosphate more indented aInd often horseshoe shaped, and(I the and p-nitro-catechol sulfate, were obtained from Sigma Chemi- cl'romilatinvwas mo(lerately conclen se(l. (b) Peroxi(lase cal Co., St. Louiis, Mo., an(l 3,3'-diaminobenzidline tetra- reAactioni productt disappeared f'romil the RER and Golgi hydrochloride vas stipplied by either Sigma Chemiiical Co. cisterinae, inidicatinig that synithesis of the enizymiie had or ICN Nuitritional Biochemicals Div., Internationial C(hemical & Nuclear Corp., Cleveland, Ohio. Gluicose oxidase (A-grade) cease(d (Table II). (c) Two populations of' graniuiles was purchase(d from Calbiochemil, San Diego, Calif. were usuially presenit, one peroxiclase-positive anid the Collection of tissues. Techini(qtues for obtaining stuspen- other peroxiclase-negative. These cutltuired monocytes sionls of Ibone marrow cells and(l eiltuiring them in a li(quiid (Fig. 2) differed f'rom typical 1lood10 monocvytes in that medium have been describecl (1 1). Specimens were harvested they contained f`ewer graniuiles. In at 2, 7, andl 14 days. peroxiclase-negative Fixatiou). Cells processecl for enzyme localization wcere addition, secondary lvsosomes fille(d with peroxidlase fixed in 1.5% (listilled gltutaraldlehydle in 0.1 M sodlitinu reactioni producit were ob)served (arrov, Fig. 2), reflect- cacodylate-HCI buffer (pH 7.4) containing 1% stucrose at ing partial clegrantilation of the peroxidase-positive 4°C for 10-30 min. storage granuiles. In other monotcytes (Fig. 3), we Enzyme procedures. After fixation, cells were vashed three times in sodiuim cacodylate-HCl buffer (pH 7.4) with commonly encouinteredl heteroplaigy of' cells such as 7% sucrose. They wvere theni incubated at 22°C inl each of neuitrophils anid . the followinig enizyme media containiing 5% suicrose: (a) Macrophage.s iitl p)eroxidase-p)ositive gi'-a nules. Graham and Karnovsky's medium for peroxidase (13), pH 7.4, By 7 (lays in cuiltiure, the milore typical feattiures of for 1 h (Figs. 1-3); (b) Tice's medium for peroxidase (14), using mcaerophages emlergedl as follows: (a) there was a the glucose oxidase metho(d for the generation of H202, for 2 h at 22°C (Figs. 4-8); (c) modified Gomori's imiedium for mairke(d inerease in cellilar size which was mainily acid phosphatase (15), pH 5.0, for 90 min (Figs. 9 and 1OA); attril)utal)le to the auigmienited cytoplasm; (b) an in- anid (d) Goldfischer's mediuimil for arylstilfatase (16), pH 5.5, crease in the number of' militochond(lria, expanision of'

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FIGuRE 1 Promonocyte from a 2-day culture reacted for peroxidase, which can be seen as a fluocculent density in the RER (rer), perinuclear cisterna (pn), and all granules (p+g). The large nucleus (N) contains a large nucleolus (nu) and heterochromatin is sparse. A few mitochondria (m) are visible, and several cytoplasmic extensions (e) extrude from the cell surface. (x 14,000.) the RER, an enlarged Golgi region with numerous into enormous cells with a host of storage granules. vesicles, and microfilaments (100 A) were also Some contained relatively few digestive vacuoles, apparent (Fig. 4); (c) characteristically, the plasma whereas, in others, these secondary lysosomes were membrane had many narrow extensions. The only plentiful (Fig. 4, inset). peroxidase-positive organielles were the granules Macrophages lacking peroxidase -single macro- formed earlier, during the promonocyte stage, and phages. By 14 days, the dominant cell type was the secondary lysosomes. Althouigh they were less promi- large macrophage without endogenous peroxidase nent at 14 days, macrophages with peroxidase-con- (Fig. 5 [Figs. 5-8 illustrate macrophages from 7- and taining granules persisted and somiietimes developed 14-day cultures lacking peroxidase.]). Whereas some

Human Macrophage Differentiation In Vitro 1557 TABLE I branes and cell sturfaces was evident (Figs. 6B and 7). Stages of Monontclear Phagocqlte Differenitiation This contact resembled the septate-like zone of ad- in Liquid Cuiltture at Vhariou.s l'imeSs hesion described in other cell types (19), but it has Davs in cudltre not been observed before between normal macro- phages. In addition to cluisters of lipid-filled macro- 2 7 14 phages with indistinct cell membranes (Fig. 8), we Promonocyte ++ + rare encouniitered conspictuouis multinuicleated giant cells Monocyte ++ + rare (not illulstrate(l). Macrophage (a) Peroxidase-positive rare ++ + Localization enzymes in (b) Peroxidase-negative rare + + + of lylsosomal 7- and Epithelioid cells 0 + + + 14-dayj culttres Giant cells () 0 + Marrow f'rom two sulbjects was cultured for 7 and 14 days, and the cells in li(quiid suspension were aspirated, fixed, and processed separately from the macrophages were free ini the liquiiid culture (Fig. 5), cells either adherenit to or loosely settled upon the many formed cluimlps (Fig. lOB) or hald become ad- suirface of the membrane. Acid phosphatase distribu- herent to the membrane (Fig. IOA). tion followed two different patterns: in both suspended Epithelioid cells. These tightly grouiped cells had and adherent cells, we located -25% that contained developecl elaborate cytoplasmic projections (Fig. 6A), reaction produict within the entire RER and in diges- similar to those described in immatuire epithelioid tive vacuioles (Fig. 9 [Figs. 9 and 10 depict 7- and 14- cells (7, 18). Occasionallv, the interdigitating pro- (lay ctulttured bone marrow cells which have been re- cesses of two adjacent macroplhages had made intimate acted for lysosomal enzymes.]). These cells were not contact, an(l a modificationi of the two p)lasmna menm- ordinarilv in close association with other macrophages.

TABLE II Ultrastr,uctural Characteristics of Various Stages of Motlononelear Phagocyte Differentiation in Cultuire

Peroxidase localization Nuiclear/ Golgi Secondarv cytoplasmic Cell type Cell size RER complex Grantiles lvsosomes ratio Ntcletis

Promonocyte (Fig. 1) 10-15 + + + - <1 (a) Round; indentations (b) Minimally condensed chromatin; large nucleoli Monocyte (Figs. 2 and 3) 8-10 - _ + + I to >1 (a) Variable shape (b) Heterochromatic, occasional nucleoli Macrophage (a) Peroxidase-positive 10-20 - - + + >1 (a) Usually eccentric; oval or (Fig. 4) indented (b) Chromatin variable; may be very condensed or euichromatic (b) Peroxidase-negative 10-20 - - - - >1 (a) Eccentric (Fig. 5) (b) Euichromatic, with large nuicleoli Epithelioid cell (a) Immature (Fig. 6) 15-20 - - - - >1 (a) Eccentric; indented (b) Matuire (Fig. 8) 15-20 - >1 (b) Euichromatic, with nucleoli >25 - - - - >1 (a) Mtultiple; randomly dispersed (b) Euichromatic, with large nucleoli

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FIGURE 2 Monocyte from a 7-day cuiltuire. Peroxidase is present only in cytoplasmic granules (p+g) and in one large vacuole (arrow)-presumably a secondary lysosome-in which degranula- tion has occurred. The many other vacuoles (v) and vesicles (ve) contain no peroxidase. Two peroxidase-negative granules can also he discerned (p-g). In the upper left-hand corner, note the portion of a promonocyte (P) with peroxidase in the RER (rer) and granules (p+g). (x 15,000.)

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FIGURE 3 Monocyte (or early macrophage) from a 7-day culture, demiionistrationi heterophagy of other leukocytes. Note the many digestive vacuoles (dv) or secondary lysosomes filled with cellular debris. Obviouis eosinoplhil crystals (arrows) caII be idleitifie(l wvithin several of these vacuoles. Some smaller peroxidase-positive granules (p*g) are still present. Cvtoplasmnic ex- tensions (e) on this cell outnuimhber those of the more typical moinocyte (lep)icted in Fig. 2. (x 13,000.)

In the seconid pattern of distribution, weak reactioni DISCUSSION prodctet wvas onily seen in some Golgi cisternae, anid mainly in large digestive vacuioles. As is apparenit in Monlocytes, uniilike nietitrophilic leukocytes, are niot Fig. IOA, some of' these mlacrophages adhered to the fUlly, difterenitiated( when releasedcl f'romi the bone miiar- dialysis membrane in comiipact nmasses. Cells found in ro\w. After cireculatinig in the bloo1( they eniter the eluLmps in the li(quid suspensioni medium also fol- tissuies, where they imiatture ilntO large, lonig-lived lowevd this latter distribution. Reaction product for aryl- macrophalges (1-3). The imiatturation of' rodent miiacro- sulf;atase wlas less extensive than that for aci(d phospha- phages lias been extensively investigated both in vitro tase in that it was rarely seen- in the RER or Golgi and in vivo (22-25). Cohni anid his co-workers (26-28) comlplex. Rather, it was observedimost ofteni in simiall have shown that smiiall, i in miatture imiacrophages develop vesicles anlle large seconidary lysosoimies (Fig. 1(B). inlto verv large phagocvtes with sizable nIticlei, plenlti-

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FIGURE 4 Large macrophage from a 14-day culture containing many peroxidase-positive grannies (p+g). m, mitochondrion; mf, microfilament; rer, scattered RER. A large peroxidase- positive inclusion is designated by an arrow. Note the slender cytoplasmic extensions (6) of an adjacent macrophage which partially interdigitate with the extensions (e) of the other cell. The inset shows two secondary lysosomes (arrows) from another macrophage, which contains whorls of membrane (w), two granules (E), and other debris. A lipid droplet is also present. ( x 12,OOO; inset x14,OOO.)

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FIGURE 5 Peroxidase-negative iniacrophage f'rom at 14-clay cuiltuire. Althiough this type ofnmacro- phage is also observed at 7 days, it is the predominant type in thie 14-day cuilture. It has an eccentric nutcleuts (N) with a distincet nuicleoluis (nu1) and( cytoplasm filledi with many of the organelles described b)eiore-i.e. miitochiondria (nin); RER (rer); a large (;olgi comiplex (C") is niear the plasma miemi-branie; andi ]Lnumerouis smiall vesicles (=-600) A). Note that f(o p)eroxidase reactionl product cani he detected at this late stage of matuiration. In adldition to occasional cle-ar xacuoles (v, any incluisions wvith miodlerately dlense miatric-es are evident. Their content is uinknowni; we are presenitly designatintg thiemi as peroxidase-ntegative granutles (p-g). Also note the p)aucitN of, 1,000 A vlesicles, wvhich are uisually presenit in great num-bers in p)eritoneal mnacrophage's fixed in vivo (8). (x 19,000.)

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FIGURE 6 (A and B). High magnifications of adjiacent macrophages, presumably epithelioid cells, cultuired for 14 days and reacted for peroxidasel, illustrating the numerous finger-like extensions and cell contacts. (A) Depicts portions of cytoplasm fromn three macrophages (MI, M2, and M3); and (B) cytoplasm from two macrophages (MI and M2). At four sites (arrows), the plasma mem- branes of the two cells in (B) are in close conitact and mianifest a structuiral modification which is better seen in Fig. 7-a higher magniification of two adjacent miembranes. These macrophages represent early or immnature epithelioid cells, containing many lipid droplets (L) which non- specifically bind diaminobenzidine, thus accounting for their dark rims. Multiple mitochondria (nin) and 100 A microfilaments (mf) are present. ((A) x21,000; (B) x28,500.) lymphoblasts, and plasma cells. Although the granulo- cytic (neutrophilic, eosinophilic, and basophilic) are also peroxidase positive, their storage granules differ sufficiently in fine structure to make them distinguishable from promonocytes (9, 33, 34). Hence, with van Furth and Fedorko (22), we support the conclusion that mononuclear phagoeytes do not develop from immature but represent a separate line of differentiation. In this in vitro system, we demonstrated that peroxidase is synthesized early, in the promonocyte stage, and packaged into storage granules by the same pathway (RER -* Golgi complex via vesicles-- granules) which has been defined for other secretory proteins (35). Monocytes did not synthesize the enzyme but contained it in their storage granules.2 With the endocytosis of dying cells, these granules degranulated; and, within 7 to 14 days, the cells became peroxidase-negative macrophages. seems to be a major cause of the loss of storage granules in the liquid culture system. How- ever, some cells retain such peroxidase-positive granules in great quantities, even after 14 days of in vitro culture. Nevertheless, the vast number of macro- phages are peroxidase-negative by 14 days, and epithe- lioid and giant cells, also all negative for peroxidase, are frequently observed. The lipid-filled clusters of epithelioid cells seen in Fig. 8 may correspond to the "giant fat" cells recently described by Dexter et al. (37) in the adherent cells cultivated from mouse bone marrow.

jp ..... '. :... . W .1 , ...... V i:.... -, , I. ... Macrophages tended to form clusters, with extensive FIGURE 7 A septate-like zone of adhesion with characteristic interdigitation between cells. Such groups have pre- spacing between two macrophages. The space between the viously been noted in animals (5, 7, 18, 38) and man two plasma membranes (arrows) measures more than 200 A (39-41) and referred to as "immature epithelioid cells" and contains irregularly spaced extracellular particles (p). The (7, 18). We found in this study that some of the cyto- intracellular face exhibits fine filaments (f). (x 100,000.) plasmic extensions had modified membrane contacts resembling septate junctions. This membrane modifica- ful cytoplasm, numerous Golgi complexes, lysosomes, tion of the mononuclear phagocyte was similar to that stacks of smooth and rough endoplasmic reticulum, described earlier by Friend and Gilula (19) in the rat and,,;. many mitochondria. These structural evolutionary adrenal cortex and in other steroid hormone-secreting changes are associated with increases in the cellular cells. Along this distinctive cell contact (19), the mem- content of protein, RNA, oxidative enzymes, and branes of apposing cells are separated by 210-300 A lysosomal hydrolases. More limited electron micro- and bisected by irregularly spaced 100-150-A extra- scopic and cyto-chemical studies have been conducted cellular particles. In freeze-fracture replicas, the cell with human mononuclear phagocytes obtained from membranes in the area of the septate contact is no dif- bone marrow (29, 30) and blood (31, 32), with soft ferent from non-junctional areas of membrane (19). Some- agar (29, 31, 32) or methylcellulose (30). what similar membrane modifications have been de- In this study, we used a liquid culture system and scribed by Sanel and Serpick (42) in leukemic monocytes, electron microscopy and cytochemistry to facilitate by Daniel and Flandrin (43) in hairy cells undergoing the detailed characterization of cellular organelles and enzyme contents of human bone marrow-derived mononuclear phagocytes. The peroxidase technique is 2 During studies on blood monocytes in vitro, Bodel et al. (36) observed that peroxidase activity rapidly appeared in the useful for identifying promonocytes, because they are RER and perinuclear cisterna within 2 h after monocyte ad- reactive for this enzyme and thus, can be distinguished herence to a fibrin-coated surface but disappeared after the from peroxidase-negative cells such as erythroblasts, cells had been cultured for 1-2 days.

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FIGURE 8 A group of large peroxidase-negative macrophages from a 14-day culture, with a viev of the nuclei of five cells (N1-N5). It is difficult to distinguish the cell borders (arrows)-a situation which has been noted previouisly in regard to mature "epithelioid" macrophages (18). Numerou1s lipid droplets (L), some with very dense borders (L'), as well as mitochondria, many vesicles, clefts (C) (20), and several Golgi conmplexes (G) are present. Here, the inset reveals intracelluilar membranes, including a Golgi region (G), at a higher inagnification. The vorm-like struetures (wo) have been demonstrated in guinea pig macrophages (21) and are thought to represent plasma membranie modifications. (x 10,000; inset x 11,000.)

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s t^. Ass;' . f t ..''~T FIGURE 9 A macrophage growing in suspension in a 7-day culture anld reacted f{r acid Phos- phatase. The entire RER (rer), excluding the perinuclear cisterna (pn), conitains moderate amounts of lead phosphate. In addition, this lysosomal enizymiie appears in imost Golgi cisterniae (Ge) as well as in somehdigestive~~~-vacuoles (dv). (x23,000.) phagocytosis, anid by Bretoni-Gorius et al. (44) in ab- here. Moreover, this iimemiibranie miiodlification differred niormal erythroblasts. To our knowledge, this is the considerably from the cell coats of adjacent imiatrophages first report of septate-like zones of attachmiient in normnal depicted by Brederoo and Daems (21), who (described macrophages, and we believe that they may serve as a spaciing of'630 A, muivch thicker thani ouir 300 A contact. adhesive elements in these cells. "Close junctions" Another findinig of this research wats the observation hiave been observed in cultured imiurine imiacrophages that adherent mlacrophages can pierce the dialysis miemii- (45) anid as so-called "junctional formiiations in im(ouse- brane, as demllonlstrated in F'ig. IOA. This imiay be an im- spleeni cultures (46), but the imorphology of these imiemil- portant discovery, because som11e investigators arc de- brane contacts is (1uite dissimiiilar from that illustrated signinlg experimenits anid interpretinig datat based oni the

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NN; FIGURE 10 (A) Adherent macrophages (M'-M2) growing on the membrane in a 7-day culture and reacted for acid phosphatase. Only scant amounts of lead phosphate are demonstrable in some Golgi cisternae (Gc) and a few digestive vacuoles (dv). Note the close cell-to-cell contact. One process has actually pierced the dialysis membrane (DM) (right corner). N, nucleus; rer, RER; m, mitochondrion. B: Three closely adjoining macrophages (M'-M3) from the liquid medium of a 14-day culture which were reacted for another lysosomal enzyme-arylsulfatase. Only the cytoplasmic vacuoles (arrows) contain reaction product. Note the many plasma membrane ex- tensions (e); such cells have been called immature epithelioid cells because of their dense packing and numerous extensions. N, nucleus. ([A] x13,000; [B] x9,500.) assumption that only small dialyzable molecules can properties of macrophage polykaryons (inflammatory giant such membranes. cells). J. Pathol. (Louid.). 113: 1 -19. penetrate 7. Adams, D. 0. 1974. The structure of mononuclear phago- A further pertinent point is the unresolved question cytes differentiating in vivo. I. Seqiuential fine and his- of why some macrophages, usually single cells, are tologic studies of' the effect of' bacilluts Calmette-Guerin actively synthesizing acid phosphatase, whereas macro- (BCG). Am. j. Pathol. 76: 17-40. phages in clusters seem to contain low levels of the 8. Nichols, B. A., 1). F. Bainton, and M. G. Farquhar. 1971. Differentiation of monocytes. Origin, nature, and fate of' enzyme. Perhaps the individual cells are better phago- their azurophil granules. J. Cell. Biol. 50: 498-515. cytes, and, having ingested a "meal," are induced to 9. Nichols, B. A., (and D. F. Bainton. 1973. Differentiation of synthesize lysosomal enzymes, as documented by Axline humllan moniocytes in bone miarrow and blood. Sequential and Cohn (47). Alternatively, the phagocytic capacity formation of'tw\o grainutle poptulations. Lab. lItvest. 29: 27- of cellular aggregates may be less than that of relatively 40. 10. Nichols, B. A., an(d 1. F. Baintoni. 1975. Ultrastructure and free macrophages. In this connection, Spector and cytocheinistrv of monionuiclear phagocytes. In Mononu- Mariano (48) found that epithelioid cells phagocytize clear Phagocytes in Immunnity, Inf'ection and Pathology. fewer latex beads and bacteria than do free macrophages. R. van Furth, editor. Blackwell Scientific Publications In conclusion, it should be re-emphasized that these Ltd., Oxford. 17-55. 11. Golde, D. WV., anid M. J. C'line. 1973. Growth of humlan observations on the localization of peroxidase were de- bone miarrowv in liquiiid cutltuire. Blood. 41: 45-57. rived from macrophages in culture. As far as we know, 12. Willcox, NM. B., D. XW. Golde, and NI. J. Cline. 1976. Cyto- similar electron microscopic and cytochemical studies chemical reactions ofhutian hematopoietic cells in li(quid of human macrophages in various tissues have not been culture. J. Ilistochieit. Cytochoem. 24: 979-983. In animal experiments, certain tissue macro- 13. Graham, R. C., and NI. J. Karnovsky. 1966. The early stages attempted. of' absorption of injected horseradish peroxidase in the phages have been found to contain peroxidase, cyto- proximal tubules of nmouse kidtney: Ultrastructural cyto- chemically identifiable in the RER and perinuclear cis- chemistry by a new technique.j. Histochem. Cy,tochem. tema. This enzyme reactivity in the RER is characteristic 14: 291-302. of resident peritoneal macrophages of the rat (49), guinea 14. Tice, L. W. 1974. Ef'fects of hypophysectomy and TSH replacemnent on the ultrastructural localization of thyro- pig (50, 51), rabbit (36), and mouse (52), as well as peroxidase. F ndocrinology. 95: 421 -433. Kupffer cells (53) and medullary lymph node macro- 15. Barka, T., and P. J. Andersoni. 1962. Hlistochemical meth- phages (54) of the rat. Such reactivity has not yet been ods for acid phosphatase using hexazonium pararosanilin reported in the tissue macrophages of man (55), but as coupler. J. Ifistocheml. Cytochem. 10: 741-753. be 16. Goldfischel-, S. 1965. The cytochemical demonstration of this point should fuirther explored. lysosomal aryl sulftatase activity by light and electron microscopy. j. Ilistoclcm. Cytochemn. 13: 520-523. ACKNOWLE DG MEN TS 17. Holtzman, E., and( R. Dominitz. 1968. Cytochemical studies We thank Ivy Hsieh and Yvonne Jac(ques for their excellent of lysosonmes, Golgi apparatus and endoplasmic reticulum technical assistance and Rosamond Michael for her helpful in and proteini uptake by adrenal medulla cells editing. of the rat. j. Histochlenm. Ci tochem. 16: 320-336. This study was supported by U. S. Public Health Service 18. Adams, D. 0. 1976. 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