Home | Development - Role of colony stimulating factor-1 in ......Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development

INTRODUCTION

Colony stimulating factor-1 (CSF-1) regulates mononuclearphagocytic cells (Stanley et al., 1983; Tushinski et al., 1982)via a high affinity cell surface receptor which is the c-

fmsproto-oncogene product (Sherr et al., 1985). Although derivedfrom a common precursor cell (reviewed by Van Furth, 1992),the mononuclear phagocytic system (MPS) is composed of acell population that is heterogeneous in terms of its tissue local-ization (Gordon, 1988; Morris et al., 1991b), functional activity

(Gordon, 1988) and expression of surface molecules (De Jong,1990). One mechanism contributing to this heterogeneitywithin the MPS may be the exposure of cells to different tissuemicroenvironments, due to differences in the local productionof growth factors, such as granulocyte-macrophage colonystimulating factor (GM-CSF) (Witmer-Pack et al., 1987;Kaplan et al., 1992) and CSF-1 (Bartocci et al., 1986; Pollardet al., 1987). CSF-1 can be locally presented either in a bio-logically active, membrane-spanning form on the surface of thecells that synthesize it (Rettenmier et al., 1987; Price et al.,

1357Development 120, 1357-1372 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

Colony stimulating factor-1 (CSF-1) regulates the survival,proliferation and differentiation of mononuclear phago-cytes. The osteopetrotic (

op/op) mutant mouse is devoid ofCSF-1 due to an inactivating mutation in the CSF-1 geneand is deficient in several mononuclear phagocyte subpop-ulations. To analyze more fully the requirement for CSF-1in the establishment and maintenance of mononuclearphagocytes, the postnatal development of cells bearing themacrophage marker antigens F4/80 and MOMA-1, in op/opmice and their normal (+/op or +/+) littermates, werestudied during the first three months of life. In normalmice, maximum expression of tissue F4/80+ cells wasgenerally correlated with the period of maximum organo-genesis and/or cell turnover. Depending on the tissue, theF4/80+ cell density either decreased, transiently increasedor gradually increased with age. In op/op mice, tissues thatnormally contain F4/80+ cells could be classified into thosein which F4/80+ cells were absent and those in which theF4/80+ cell densities were either reduced, normal orinitially normal then subsequently reduced. To assesswhich F4/80+ populations were regulated by circulatingCSF-1 in normal mice, op/op mice in which the circulatingCSF-1 concentration was restored to above normal levelsby daily subcutaneous injection of human recombinantCSF-1 from day 3 were analyzed. These studies suggestthat circulating CSF-1 exclusively regulates both theF4/80+ cells in the liver, spleen and kidney and the MOMA-1+ metallophilic macrophages in the spleen. Macrophages

of the dermis, bladder, bone marrow and salivary gland,together with a subpopulation in the gut, were partiallyrestored by circulating CSF-1, whereas macrophages of themuscle, tendon, periosteum, synovial membrane, adrenalsand the macrophages intimately associated with theepithelia of the digestive tract, were not corrected byrestoration of circulating CSF-1, suggesting that they areexclusively locally regulated by this growth factor. Langer-hans cells, bone marrow monocytes and macrophages ofthe thymus and lymph nodes were not significantly affectedby circulating CSF-1 nor decreased in op/op mice, consis-tent with their regulation by other growth factors. Theseresults indicate that important differences exist amongmononuclear phagocytes in their dependency on CSF-1and the way in which CSF-1 is presented to them. They alsosuggest that the prevalent role of CSF-1 is to influenceorganogenesis and tissue turnover by stimulating the pro-duction of tissue macrophages with local trophic and/orscavenger (physiological) functions. Macrophages involvedin inflammatory and immune (pathological) responsesappear to be dependent on other factors for their ontogen-esis and function. This study provides a base from whichto analyze further the mechanisms of regulation and phys-iological roles of CSF-1-dependent tissue macrophages.

Key words: Colony Stimulating Factor-1, CSF-1, macrophages,growth factor, osteopetrotic mouse, op mutation, organogenesis,tissue remodelling

SUMMARY

Role of colony stimulating factor-1 in the establishment and regulation of

tissue macrophages during postnatal development of the mouse

Marco G. Cecchini1, Melissa G. Dominguez2, Simonetta Mocci2, Antoinette Wetterwald1, Rolf Felix1, Herbert Fleisch1, Orin Chisholm2, Willy Hofstetter1, Jeffrey W. Pollard2,3 and E. Richard Stanley2,*1Department of Pathophysiology, University of Berne, CH-3010 Berne, Switzerland and the Departments of 2Developmental andMolecular Biology and 3Obstetrics and Gynecology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, New York, NY 10461, USA

*Author for correspondence

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1992) or alternatively, as the predominant secreted (proteo-glycan) form (Price et al., 1992), which may be targeted to atissue-specific extracellular matrix via its glycosaminoglycanmoiety. Differences between tissue microenvironments mayalso arise due to differences in cellular accessibility to circu-lating growth factors. For example, CSF-1 is found in the cir-culation (Stanley, 1979) and certain macrophage populations,such as Kupffer cells (Bartocci et al., 1987), have access to cir-culating CSF-1, while others, such as peritoneal macrophages(Chen, 1991), do not.

The CSF-1-less osteopetrotic (op/op) mutant mouse exhibitsimpaired bone resorption associated with a paucity of osteo-clasts (Marks and Lane, 1976), is deficient in bone marrowmacrophages, blood monocytes and serosal cavitymacrophages (Wiktor-Jedrzejczak et al., 1982, 1990; Felix etal., 1990b) due to an inactivating mutation in the coding regionof the CSF-1 gene (Wiktor-Jedrzejczak et al., 1990; Yoshidaet al., 1990; Felix et al., 1990b; Pollard et al., 1991). Admin-istration of CSF-1 to newborn op/op mice cured their osteopet-rosis and substantially corrected the deficiencies in osteoclasts,bone marrow cellularity and blood monocytes, but not the de-ficiencies of the serosal resident macrophage populations(Kodama et al., 1991; Felix et al., 1990a; Wiktor-Jedrzejczaket al., 1991), suggesting that some macrophage populations aredependent on circulating CSF-1, while others require locallyproduced CSF-1. Thus, the op/op mouse is an ideal model inwhich to investigate how CSF-1 regulates the anatomical dis-tribution of the MPS. Indeed, preliminary descriptions of howthis mutation affects the distribution and morphology ofmacrophages detected by the expression of the macrophagespecific antigen, F4/80 (Austyn and Gordon, 1981), indicatedthat their number was reduced in the majority of tissuesexamined in the adult op/op mouse (Naito et al., 1991; Wiktor-Jedrzejczak et al., 1992b). Furthermore, these cells generallyexhibited a morphology consistent with their incompletedifferentiation.

We here describe the distribution of tissue macrophages innormal (+/+ or +/op) and mutant (op/op) mice during the firstthree months of postnatal development. Furthermore, we haveinvestigated the role of circulating CSF-1 in tissue mononu-clear phagocyte regulation by analysis of the redistribution oftissue macrophages in op/op mice when circulating CSF-1levels are restored during this period.

MATERIALS AND METHODS

AnimalsOsteopetrotic op/op mice and littermate controls (+/+ or +/op) werebred and maintained in isolated units of the Albert Einstein Collegeof Medicine animal house and of the Department of Pathophysiologyat the University of Berne as described previously (Wiktor-Jedrzejczaket al., 1990; Felix et al., 1990b; Pollard et al., 1991). Mice were fedad libitum with powdered chow and infant milk formula (Enfamil).At birth, the op/op homozygotes were radiologically distinguishedfrom the phenotypically normal siblings based on the dense aspect ofbone due to the absence of a distinct medullary cavity (Felix et al.,1990a). At 10 days of age, they were distinguished by the absence ofincisors and by a domed skull (Marks and Lane, 1976). Three month-old op/op mice weigh 65% as much as littermate control mice.

CSF-1 treatmentHighly purified recombinant human CSF-1, a generous gift from

Chiron Corporation (Emeryville, California), was suspended in phys-iological saline (2

×107 units, equivalent to 0.24 mg protein/m; Stanleyet al., 1972) and stored at −20°C. Unless otherwise indicated, theop/op mice were subcutaneously inoculated with 50 µl of this solution(106 units per mouse) daily from 3 days of age. Littermate controlmice were injected daily with 50 µl of physiological saline. Mice werekilled at 3-4 months of age.

ImmunohistochemistryFor immunostaining with the macrophage specific rat monoclonalantibody F4/80 (Austyn and Gordon, 1981), op/op mutants and theirnormal siblings at the age of 2 days, 2 weeks, 2 months and 3 monthsand the CSF-1 injected op/op mice were perfused in vivo under etheranesthesia through the left ventricle (Hume and Gordon, 1983) withperiodate-lysine-2% paraformaldehyde-0.05% glutaraldehyde, pH 7.4(PLPG) (McLean and Nakane, 1974). Tissues were then excised andfixed for 6 hours at 4°C in the same fixative. The tibia, including theknee-joint, was decalcified for 48-72 hours in several changes ofacid/citrate buffer (13% sodium citrate in 2% formaldehyde, pH 4.7with formic acid) (Hume et al., 1984). The tissues were then dehy-drated and embedded in polyester wax. Sections of 5 µm were cut andair dried on gelatin coated slides.

For immunostaining with the rat monoclonal antibody MOMA-1,specific for marginal metallophilic macrophages of the spleen (Kraaland Janse, 1986), frozen sections of spleens from op/op mutant andtheir normal siblings at the age of 2 months and from CSF-1 injectedop/op mice were fixed in hexazotized pararosaniline for 1 minute asdescribed (Kraal and Janse, 1986; De Jong et al., 1991), stored at −70°C and immunostained within 1 week.

Immunostaining was performed according to the indirect peroxi-dase-conjugated streptavidin procedure (Hume and Gordon, 1983;Felix et al., 1990b), using rat gamma globulin (5 µg/ml) as a control.

Quantification of F4/80+ cells in tissue sectionsAt each age, at least two op/op and two normal mice were examined.For tissues with linearly arrayed macrophages (synovial membrane,epidermis and periosteum), F4/80+ cell densities were determinedusing an ocular micrometer and expressed in cells/mm; otherwise thedensities were determined by scoring at least 5-10 fields andexpressed in cells/mm2. In heterogeneous tissues, such as bonemarrow, or in tissues with an uneven parenchymal distribution ofF4/80+ cells, such as spleen and lymph node, the F4/80+ cell densitywas determined with the aid of the Zeiss I integrating eyepiece(Meunier and Courpron, 1973) and expressed in cells/mm2 of thespecific tissue of interest. F4/80+ cell densities were averages derivedfrom 2 animals and standard deviations for multiple counts (n>5) were<10% of the means. Most tissues were examined in the two partici-pating institutions and the results independently verified.

RESULTS

Normal postnatal development of tissuemacrophagesSections of tissues obtained from 2-day- to 3-month-old phe-notypically normal (+/+ or +/op) mice were immunostained forF4/80 antigen in order to follow the postnatal development oftheir macrophage populations. Tissues have been classifiedinto groups (Table 1) depending on the behavior of theirF4/80+ populations in order to simplify the description and dis-cussion of results. Here and subsequently in the results section,the text refers only to the topology and morphology of stainedcells. The reader is referred to the tables for the changes in celldensity.

M. G. Cecchini and others

1359CSF-1 regulation of tissue macrophages

(a) Tissues in which the density of F4/80+ cellsdecreases with ageIn muscle and tendon, macrophages occur in regions of tissueremodeling, for example in areas of osteoinsertion andgenerally they are spindle-shaped with the major axis parallelto the fibers of both muscle and tendon. Macrophages of thedermis are generally round, except those in proximity to thebasal epidermal layer, which are aligned with it as spindle-shaped cells. This is particularly evident at 2 days of age (Fig.1A). At 2 weeks of age they are sometimes found to surroundthe developing hair bulbs. The F4/80+ cells in neonatal liver(Kupffer cells) are evenly distributed within the liverparenchyma. They are highly dendritic and intimately associ-ated with islands of hematopoietic cells (Fig. 2A). By 2months, their density is dramatically decreased in the cen-trilobular regions but maintained around the portal triads andthey are much less dendritic (Fig. 2C). By this time,hematopoietic cells are no longer visible. At 2 days of age,dendritic F4/80+ cells in the retina, with characteristics ofmicroglia, are restricted to the inner nuclear layer and to thedeveloping inner plexiform layer (Fig. 3A), as previously

reported (Hume et al., 1983a). By 2 weeks these cells areabsent from the above sites, but similar dendritic cells appearin the outer plexiform layer. At 2 and 4 months there are nodetectable F4/80+ cells in the retina.

(b) Tissues in which the density of F4/80+ cellstransiently increases in the early postnatal periodAt 2 days of age, F4/80+ cells in the stomach are exclusivelylocated in the subglandular region of the lamina propria (notshown). They are few in number, but by 14 days their densityis greatly increased in this region, they are more closely asso-ciated with the chief cells and they are much more dendritic.F4/80+ cells are also apparent along the glandular columns inassociation with parietal and mucous cells. In addition, by 14days they are present in the lamina propria of the non-glandularregions and at a much lower density in the muscularis externa.At 2 and 4 months, the distribution and morphology of theF4/80+ cells is essentially unchanged. At 2 days of age, theF4/80+ cells of the small intestine are characteristicallyrounded and present predominantly in the lamina propriatowards the base of the villi (Fig. 4A). Their density in thisregion increases dramatically over the ensuing 12 days and thecells adopt a more spindle-shaped morphology. At later agesthey are predominantly found within the villi, closely associ-ated with the capillaries and the base of the columnar cell layer.Occasional cells are also found lining the inner surface of theserosa (Fig. 4C). Similar postnatal changes in the morphologyand distribution of F4/80+ cells are observed in the largeintestine, where the cells are also located in the lamina propria.

At 2 days of age, lymph node F4/80+ cells are uniformlydistributed throughout the still primitive parenchymalstructure. At 2 weeks, F4/80+ cells are present in both thecortex and the medulla. They are abundant adjacent to the sub-capsular sinus and concentrated within the medullary sinuses.The cells are stellate with some scattered reticularmacrophages in the germinal centers of the lymphoid follicles.By two months, the number of F4/80+ cells is decreased, espe-cially in the subcapsular area. However, they are still well rep-resented in the medullary region. F4/80+ cells in the 2-daythymus are found predominantly in the cortical area, wherethey are slightly spread and surround developing thymocytes.Less spread F4/80+ cells are also found, more sparsely dis-tributed, in the medulla. By 2 weeks, slightly dendritic F4/80+cells can be seen along the connective tissue septae associatedwith the arterioles and around the corticomedullary junction.Scattered, less dendritic, F4/80+ cells are also apparent in themedulla and surrounding the Hassel’s corpuscles. By 2months, the number of positive cells is decreased, especiallyin the subcapsular region. They are still well represented in themedulla.

Within the first 2 weeks the F4/80+ cells in periosteum areassociated with sites of active bone modeling during rapid bonegrowth. Subsequently, they are predominantly found at themetaphyseal junction. As described by others, the elongatedF4/80+ cells of the kidney are more dense in the medulla andprimarily line the medullary and cortical tubules (Hume andGordon, 1983) and in cross sections of the skin, the Langer-hans cells in the epidermis are elongated and intercalated withcells of the basal layer (Hume et al., 1983b, Fig. 1).

Two morphologically distinct F4/80+ cell types wereresolved in the bone marrow. The monocyte type are lightly

Table 1. Relative changes in tissue F4/80+ cell densityduring postnatal development in normal (+/+ or +/op)

miceAge

2 2 2 3Tissue days weeks months months

(a) Decreasing F4/80+ cell density

Muscle, striated 100 *(91)** 10 0 0Tendon 100 (619) 35 8 0Dermis 100 (509) 46 21 23Liver 100 (515) 85 60 45Retina †† †† †† ††

(b) Transiently increasing F4/80+ cell density

Stomach 5 100 (350) 85 75Small intestine 8 100 (205) 80 70Large intestine 5 100 (266) 85 70Lymph node †† 100 (512) 64 55Thymus 51 100 (412) 70 45Periosteum 36 100 (9)† 78 64Kidney 46 100 (198) 70 48Epidermis, 57 100 (26)† 81 87

Langerhans cellsBone marrow, 67 100 (916) 78 84

‘macrophage’

(c) Increasing F4/80+ cell density

Bladder †† 29 64 100 (197)Spleen, red pulp 15 65 82 100 (792)Adrenals †† 71 78 100 (85)Salivary gland, †† 32 100 (137) 93

submandibularSalivary gland, †† 21 85 100 (124)

sublingualSynovium, 64 84 100 (52)† 78

‘type A cells’Bone marrow, <5 72 91 100 (1185)

‘monocyte’

*Percentage of the maximum cell density for each tissue.**Densities in cells/mm2 or †cells/mm, average for at least 2 mice.††Tissue too small or F4/80+ cell density too low to provide a reliable

figure.

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stained, rounded cells whereas the macrophage type, describedby Hume as resident bone marrow macrophages (Hume et al.,1983b), stain heavily and are highly dendritic (Fig. 5). At 2days of age, they are smaller and less dendritic than in oldermice (Fig. 5A). By 14 days of age, they assumed the typicaladult stellate morphology (Felix et al., 1990b) (Fig. 5B). Thesecells are present in the diaphysis and penetrate the primaryspongiosa to the level of the capillary invasion front where they

assume a spindle shape and are spread on the outer walls ofthe vessels.

(c) Tissues in which the density of F4/80+ cellsincreases with ageF4/80+ cells are almost absent from 2-day bladder. By 14 days,they are found as spindle-shaped cells in the lamina propria,occasionally in close relationship to the basal cell layer of the


Fig. 1. Epidermis and dermis (transverse section). Immunostaining with monoclonal antibody F4/80; (A,C) normal littermates (+/+ or +/op);(B,D) op/op mutants. (A,B) 2-day-old scalp; (C,D) 2-month-old ear. (F) Ears of a 3-month-old, CSF-1-injected op/op mouse and E, its age-matched normal littermate. e, epidermis; d, dermis; c, cartilage. Langerhans cells (arrows) and dermal macrophages (arrowheads). Bar, 50 µm.Not counterstained.


transitional epithelium. They are also found scattered withinthe smooth muscle layer. By 2 months of age, their density inboth locations is substantially increased. Significantly, the cellswithin the lamina propria acquire a much closer relationshipwith the basal cell layer and are much more dendritic. SomeF4/80+ cells are associated with the capillaries and extendprocesses into the epithelium. By 4 months of age, the vastmajority of F4/80+ cells are associated with the basal layer ofthe lamina propria while their density in the smooth musclelayer is reduced.

In the spleen, F4/80+ cells are initially rounded (day 2) butby 2 weeks they become stellate. They are exclusively located

in the red pulp where they represent the major sub-populationof splenic macrophages (De Jong, 1990). The F4/80+ cells ofthe adrenal gland at 2 days, prior to the development of thespecialized architecture of this gland are round and randomlydistributed in the parenchyma. At 2 weeks, they are stellate andpreferentially located in the outer cortex. By 2 months, theirnumbers have increased in the outer cortex and they are alsofound in the inner cortex and in the periphery of the medulla.In the inner cortex, F4/80+ cells are smaller than in the outercortex, while in the medulla they assume a more spindledshape.

The F4/80+ cells of the submandibular and sublingual

Fig. 2. Liver (frontal section). Immunostaining withmonoclonal antibody F4/80. (A,C) Normal littermates (+/+ or+/op); (B,D) op/op mutants. (A,B) 2 days old; (C,D) 3 monthsold; (E) 3-month-old CSF-1-injected op/op mouse. Bar, 50µm. Not counterstained.

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salivary glands are found in both subepithelial and interstitiallocations. In the interstitium they are scattered and less spread.In the submandibular gland they are closely associated with thetubuloalveoli and are spindle-shaped. A similar morphology ofF4/80+ cells is observed in sublingual salivary glands wherethey are located on the epithelial side of the basementmembrane and associated with the secretory duct.

Between 2 and 14 days of age, the density of F4/80+ cellslining part of the synovial membrane (synovial ‘A-cells’)increases significantly and remains constant thereafter (Fig. 6).At 2 days, however, many F4/80+ cells are also uniformlyscattered within the synovial cushion. By 14 days, consistentwith a redistribution from the synovial cushion to the synovialmembrane, the density of synovial cushion macrophages isdecreased. During the first 2 weeks of life, the monocytes ofthe bone marrow (Fig. 5) are mainly localized in the diaphysisand are scarce in the metaphysis, being almost totally absentwithin 300 µm of the capillary invasion front. By 2 months ofage, when longitudinal bone growth has substantially slowed,the monocytes are more evenly distributed between the meta-physeal and diaphyseal regions. In the diaphyseal region, themonocytes are more often found closely associated with thedendritic macrophages.

Effect of the

op mutation on the postnataldevelopment of tissue macrophagesA significant proportion of the developmental changes in themouse mononuclear phagocytic system occurs postnatally (seeabove and Morris et al., 1991b). To determine the role of CSF-1 in this process, the postnatal changes in F4/80+ cells in theCSF-1-less op/op mutant were compared with those describedabove for normal littermate control mice (Tables 1 and 2).Tissues were divided into the groups a-d (below), according tothe behavior of their F4/80+ populations in op/op mice.

(a) Tissues in which F4/80+ cells are virtually absentthroughout postnatal lifeThe macrophage populations of striated muscle, tendon andkidney failed to develop in op/op mice. Although present at asmall fraction of their normal density, some macrophages of


Fig. 3. Retina (sagittal section). Immunostaining with monoclonal antibody F4/80. (A) Normal littermates (+/+ or +/op); (B) op/op mouse. 2days old. c, choroid; i, inner nuclear and plexiform layers; o, outer plexiform layer. Bar, 50 µm. Not counterstained.

Table 2. Developmental changes in tissue F4/80+ celldensity in CSF-1-less (op/op) mice

Age

2 2 2 3Tissue days weeks months months

(a) F4/80+ cells absent

Muscle, striated 0* (100)** 0 (10) 0 (0) 0 (0)Tendon 0 (100) 0 (35) 0 (8) 0 (0)Dermis 9 (100) 22 (46) 10 (21) 3 (23)Periosteum 2 (36) 9 (100) 21 (78) 18 (64)Synovium, 0 (64) 0 (84) 1 (100) 1 (77)

‘type A cells’Kidney 2 (67) 0 (100) 2 (70) 10 (48)Retina † † † †

(b) F4/80+ cells reduced

Adrenals † 9 (71) 17 (78) 26 (100)Bladder † 7 (29) 15 (64) 17 (100)Salivary gland, † 12 (32) 23 (100) 31 (93)

submandibularSalivary gland, † 5 (21) 19 (85) 36 (100)

sublingualBone marrow, 3 (46) 15 (100) 30 (78) 34 (84)

‘macrophage’

(c) F4/80+ cell initially normal

Liver 81 (100) 55 (85) 30 (60) 20 (45)Stomach 5 (5) 35 (100) 30 (85) 15 (75)Small intestine 5 (8) 30 (100) 25 (80) 10 (70)Large intestine 5 (5) 30 (100) 20 (85) 10 (70)Spleen, red pulp 11 (15) 63 (65) 48 (82) 54 (100)

(d) F4/80+ cells normal

Epidermis, 58 (58) 104 (100) 84 (82) 85 (87)Langerhans cells

Thymus 48 (51) 91 (100) 53 (70) 32 (45)Lymph node † 98 (100) 46 (64) 39 (55)Bone marrow, 5 (4) 172 (72) 93 (91) 98 (100)

‘monocyte’

*op/op cell density expressed as percentage of the maximum cell densityfor each normal tissue.

**Normal cell density expressed as percentage of the maximum celldensity for normal tissues (from Table 1).

†Tissue too small or F4/80+ cell density too low to provide reliable figure.


the dermis were always present throughout life. They werefound exclusively in the region immediately below theepidermal layer and exhibited a morphology similar to thosefound in the same region of normal mice. In contrast, themacrophages in the deeper region of the dermis were virtuallyabsent, except at 2 weeks of age, when rounded macrophageswere found in association with the developing hair bulb, as innormal mice. Interestingly, in op/op mutants this deep regionof the dermis was thinner (hypotrophic and hypoplastic) thanage-matched normal littermates at all the ages examined (Fig.1).

Although extremely reduced in density during the first 2weeks of postnatal life, some macrophages developed in the

periosteum by 2 and 3 months of age, but they were exclu-sively concentrated at the remodeling site, located at the meta-physeal-diaphyseal junction. True synovial ‘A-cells’ werealways absent and occasional F4/80+ cells could only be foundimmediately below the synovial membrane, never adjoining it.Noteworthy was the hypoplasia and, in extreme cases, theatrophy of this membrane, especially at the later agesexamined. A peculiar situation was observed in the retinawhere, in contrast to normal littermate control mice, F4/80+cells could not be detected in 2-day (Fig. 3B) or 2-week-oldop/op mice. However, by 2 months of age, dendritic F4/80+cells became apparent in the inner nuclear and plexiform layersof the retinas of op/op mice with a density matching their

Fig. 4. Small intestine (transverse section). Immunostainingwith monoclonal antibody F4/80. (A,C) Normal littermates(+/+ or +/op); (B,D) op/op mice. (A,B) 2 days old; (C,D) 3months old; (E) 3-month-old CSF-1-injected op/op mouse. v,villus; m, muscularis externa. Bar, 50 µm. A,B, notcounterstained; C,D,E, hematoxylin counterstain.

1364 M. G. Cecchini and others

Fig. 5. Bone marrow (longitudinal section of the tibia throughthe sagittal plane). Immunostaining with monoclonal antibodyF4/80. (A,C,E) Normal littermates (+/+ or +/op); (B,D,F) op/opmice. (A,B) 2 days old; (C,D) 15 days old; (E,F) 3 months old;(G) 3-month-old CSF-1-injected op/op mouse. m, marrow; t,trabecular bone; c, cortical bone; p, periosteum. Bar, 50 µm.Not counterstained.


density in 2 day control mice. They persisted with a slightlylower density at 3 months. While the appearance of these cellsin op/op mice was delayed and their presence less transient,their morphology was indistinguishable from the microglia ofthe 2-day-old control mice.

(b) Tissues in which the density of F4/80+ cells isreduced throughout postnatal lifeIn the adrenal glands, by 2 weeks, F4/80+ cells are less stellatethan in control mice and are found preferentially in the outercortex and between the cortex and medulla. The perivascularlylocated cells disappear with age. In the 2-week-old bladder,F4/80+ cells are scarce, less spindle-shaped than in control

mice and located in the lamina propria. By 2 months, comparedwith control mice, F4/80+ cells are less dendritic and not soclosely associated with the transitional epithelium. In salivaryglands, by two weeks, F4/80+ cells are flatter, less spindle-shaped and smaller than in control mice. They are more inter-stitially localized and less associated with the tubuloaveoli(submandibular glands) or secretory ducts (sublingual glands).In contrast to control mice, throughout the postnatal period inop/op mice, bone marrow macrophages are confined exclu-sively to the diaphyseal region. There was a progressiveenlargement of the marrow space with age. By 2 weeks, thepercentage of the diaphyseal region occupied by marrow was46% and this increased to 72% by 2 months of age. Within the

Fig. 6. Synovial membrane (sagittal section of the knee joint).Immunostaining with monoclonal antibody F4/80. (A,C) Normallittermates (+/+ or +/op); (B,D) op/op mice. (A,B) 2 days old;(C,D) 2 months old; (E) 3-month-old CSF-1-injected op/opmouse. s, synovial membrane; c, articular cartilage; t, tendon; m,meniscus. Bar, 50 µm. Not counterstained.

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first 2 weeks the strongly F4/80+ bone marrow op/opmacrophages were much less dendritic than those of littermatecontrol mice (Fig. 5B,D). By 2 months, only approximately50% of the F4/80+ cells had adopted the typical dendritic shapeof cells from control mice (Fig. 5F).

(c) Tissues in which the density of F4/80+ cells is initiallynormal and changes with ageIn liver, there was no apparent difference in the morphologyof the Kupffer cells at any stage but there was a precociousloss of their centrilobular localization in op/op mice (Fig.2B,D). In op/op mice, the distribution of macrophages at day2 in stomach, small (Fig. 4B) and large intestine was normal.However, at later times, in contrast to control mice, in thestomach they were virtually absent from the lamina propriasurrounding the glandular columns. In the intestine the villouscores (Fig. 4D) and the connective tissue surrounding thecrypts of Lieberkuhn were similarly devoid of cells. F4/80+cells of the gastrointestinal tracts of op/op mice tended to besmaller, more rounded and reminiscent of these populations innormal day-2 mice.

There was no significant difference in the distribution ofF4/80+ cells in the spleen. However, their morphology wasdifferent at birth and 2 weeks of age, in that cells that were lessdendritic and more rounded than in normal mice appearedunevenly distributed in the red pulp. Differences in morphol-ogy at the later time points could not be detected. In order todetect populations of splenic macrophages not recognized bythe F4/80 antibody, in particular the marginal metallophilicmacrophages (Hume et al., 1983b; Witmer and Steinman,1984), frozen sections from two-month-old op/op and controlmice were immunohistochemically stained with monoclonalantibody MOMA-1. Interestingly, the population of MOMA-1+ marginal metallophilic macrophages, found in controlsections at the junction between the red and white pulp, wastotally absent in the sections from two day and two month (Fig.7) old op/op mice.

(d) Tissues in which the density of F4/80+ cells isnormal throughout lifeNo difference in the distribution or morphology of epidermalLangerhans cells (Fig. 1) and of F4/80+ cells in thymus andlymph node could be discerned, except in the case of the bonemarrow monocytes, which tended to closely associate withinthe larger marrow spaces.

Effect of restoration of normal circulatingconcentrations of CSF-1CSF-1 is normally found (approx. 12-18 ng/ml) in the circula-tion (Wiktor-Jedrzejczak et al., 1990). In order to assesswhether macrophage populations required locally presented,rather than circulating CSF-1 for their development, circulat-ing CSF-1 in op/op mice was restored to normal levels or abovewith human recombinant CSF-1 from 3 days of age. In 8-week-old op/op mice that were injected daily as described, the con-centration of circulating human CSF-1 at 24 hours after theirlast injection was approximately the concentration of circulat-ing CSF-1 in normal control mice (18 ng/ml) (Wiktor-Jedrzejczak et al., 1990), so that on average the circulatingCSF-1 concentration in the injected mice was above normal.According to their response to injected CSF-1 (Table 3), tissue

macrophages could be classified into those that apparentlyrequired circulating CSF-1 and those that did not.

(a) Tissues in which the densities of F4/80+ cells arerestored to at least normal levelsWhile macrophage densities of spleen red pulp returned tonormal, F4/80+ cell densities in the livers (Fig. 2E) andkidneys of the CSF-1 treated op/op mice were several foldhigher than in the age-matched, littermate controls (Table 3).It was not possible to discern a difference in the morphologyof splenic F4/80+ cells from op/op mice, normal mice andinjected op/op mice at three months of age. In the liver, thecells were more concentrated around the portal triads andaround the central vein and adopted a more elongated mor-phology than in the uninjected mice. Similarly in the kidney,the F4/80+ cells were more spindle-shaped than in the unin-jected op/op mice and were concentrated in the medulla alongthe tubules and within the juxta-glomerular complex, as is thecase of normal kidney (see above and Hume and Gordon,1983). A dramatic restoration of the density and morphologyof the MOMA-1+ marginal metallophilic macrophages of thespleen in their normal location between the white and red pulpwas observed in the CSF-1 injected op/op mice (Fig. 7C). Thedensity of microglia in the inner nuclear and plexiform layersof the retinas of op/op mice was approximately twice theirdensity in age-matched op/op mice that did not receive CSF-1, and approximately 1.5 times their density in normal 2-day-


Table 3. Effect of CSF-1 injection of op/op mice on tissueF4/80+ cell densities at 3 months of age

CSF-1Effect Tissue Control injected

(a) Restoration to at Liver 45* 354least normal Kidney 21 505F4/80+ cell density Spleen, red pulp 58 91

Retina † †

(b) Partial restoration Dermis 13 85of F4/80+ cell Bladder 17 48density Salivary gland, 33 66

submandibularSalivary gland, 36 57

sublingualStomach 21 40Small intestine 15 25Large intestine 15 22Bone marrow, 40 60

‘macrophage’

(c) Unaffected F4/80+ Muscle, striated ** **cell density Tendon ** **

Periosteum 28 29Synovium, 1 2

‘type A cells’Adrenals 26 24Bone marrow, 98 88

‘monocyte’Epidermis, 97 94

Langerhans cellsThymus 73 76Lymph node 71 74

*Percentage of the F4/80+ cell density of age-matched (littermate) control(+/+ or +/op) mice.

**F4/80+ cells not detected in any group.†Tissue too small and F4/80+ cell density too low to provide reliable

figure.


old mice. These cells also had longer and more delicate pro-jections than their counterparts in uninjected mice.

(b) Tissues in which the densities of F4/80+ cells arepartially restoredIn these tissues, the F4/80+ cell densities increased from 1.5-6.5 times the densities observed in untreated op/op mice, butat best attained 85% of the densities of the age-matched, lit-termate controls (Table 3b). In the bladders of CSF-1-treatedop/op mice, the cells were found preferentially in the laminapropria surrounding the vessels and they were less spindle-shaped and less associated with the basal layer of the transi-tional epithelium than the F4/80+ cells in untreated mice. TheF4/80+ cells within the smooth muscle layer were not restoredby CSF-1 treatment. In the salivary glands, the F4/80+ cellswere more equally distributed between the glandular and non-glandular parenchyma and were smaller and less dendritic thanin untreated mice. The data in Table 3 suggesting that there ispartial restoration of F4/80+ cell densities in the stomach andintestine is misleading if one considers subpopulations withinthese tissues. While there was partial restoration of the densityof the cells in the lamina propria at the base of the mucosa, thecell density in the lamina propria surrounding the glandularcolumns of the stomach, the Crypts of Lieberkuhn and the villiwas unaffected by CSF-1 treatment (Fig. 3E). The morphologyof the F4/80+ cells at the base of the mucosa was unaffectedby CSF-1 treatment. A difference in the local distribution ofthe macrophages induced by CSF-1 was also evident in the

dermis. There was a clear reconstitution of the macrophagedensity in the deep region of the dermis, but these cells werelarger and more irregular than their normal counterparts. Incontrast, in the region immediately below the epidermis, themacrophage density was virtually unchanged compared withuntreated op/op mice. The cell density and morphology ofmacrophages of the bone marrow were both partially restoredby CSF-1 treatment. Only one-third of these cells acquired thetypical, highly dendritic morphology exhibited in normal mice.They were distributed in both diaphysis and metaphysis, but inthe metaphysis they never reached the capillary invasion front.

(c) Tissues in which the densities of F4/80+ cells are notaffected (Table 3)This group includes F4/80+ cells in striated muscle, tendon,periosteum, synovial membrane and adrenals, whose densitiesremain very low throughout postnatal to adult development inop/op mice. It also includes bone marrow monocytes, Langer-hans cells, thymus and lymph node macrophages whosedensities were approximately normal in op/op mice. In neithergroup was there significant effect of restoration of circulatingCSF-1 on the morphology or distribution of F4/80+ cells.

Effect of injected dose of CSF-1The densities of F4/80+ cells in the livers and kidneys of theCSF-1-injected mice were several-fold higher than theirdensities in age-matched control mice (Table 3), possiblyreflecting the choice of a CSF-1 dose that ensured that the

Fig. 7. Spleen (longitudinal section). Immunostaining withmonoclonal antibody MOMA-1. (A) Normal littermates (+/+ or+/op); (B) op/op mouse. 2 months old. (C) 3-month-old CSF-1-injected op/op mouse. w, white pulp; r, red pulp. Bar, 50 µm.Hematoxylin counterstain.

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injected mice possessed at least normal circulating concentra-tions of the growth factor at all times. Because of the sensitiv-ity of the macrophages in these organs to circulating CSF-1,they were chosen to examine the CSF-1 dose-response. Asshown in Table 4, a dose of 3×104 units or 0.36 µg per day wassufficient to maintain the F4/80+ cell densities found in thelivers and kidneys of normal littermates. Higher concentrationswere needed, however, to maintain the splenic population.

DISCUSSION

The postnatal development of tissue F4/80+ cells innormal miceIn general, the maximal expression of F4/80+ cells duringnormal postnatal development correlated with the period ofmaximum organogenesis and/or cell turnover. For example,muscle development peaks immediately prior to birth, and thisis reflected in the rapid postnatal decline in F4/80+ cells (Rugh,1991). On the other hand, tissues such as tendon, which issubjected to extensive remodeling as the osteoinsertionchanges with longitudinal bone growth, and dermis, whichcontinues to expand after birth, have a significantly slowerdecrease in F4/80+ cell densities. The changes observed in themicroglial population of the retina are also consistent withscavenger and trophic roles of F4/80+ cells during develop-ment. As previously noted (Hume et al., 1983a), the coinci-dence of macrophage invasion of the different layers of theretina with neuronal cell death, a characteristic of the devel-opment of central nervous tissue (Oppenheim, 1981), corrob-orates their scavenger function. In addition, their subsequentlocalization in the plexiform layers may also be ascribed totheir trophic role in promoting neurite extension (Perry et al.,1987). Consistent with the possible role of the stromalmacrophages of the hemopoietic tissues in regulatinghematopoiesis (Gordon et al., 1986; Crocker et al., 1988;Morris et al., 1991a,b), the postnatal decrease in F4/80+ cellsin liver may be related to its transition from a fetal hematopoi-etic to adult parenchymal organ.

Gut and kidney are known to undergo substantial postnataldevelopment associated with acquisition of adult function andthis is paralleled by the flux in macrophage population (Rugh,1991). Increased F4/80+ cell expression in thymus, lymphnode and epidermis (Langerhans cells) is correlated with thedevelopment of immune competence (Bier et al., 1981). Thepostnatal changes in bone marrow macrophages may be relatedto two separate physiological changes. During the first few

weeks of postnatal life there is a progressive increase in bonemarrow hematopoiesis, which is probably supported by theresident F4/80+ cell population (Gordon et al., 1986; Crockeret al., 1988; Morris et al., 1991b). Simultaneously, in con-junction with the peak in expression of periostealmacrophages, the maximum rate of longitudinal bone growth,demanding substantial bone modeling and remodeling, isachieved.

In tissues with a sustained increase in F4/80+ cells the cor-relation between expression of high F4/80+ cell density andtissue turnover is less apparent. While the function ofmacrophages within these tissues has not been studied in detail,it is clear that there is a persistently high cell turnover in adultbone marrow and spleen. The F4/80+ monocyte-like cells inthe latter two organs probably represent the precursors of bloodmonocytes (Gordon et al., 1986) for which there is a continualdemand in the adult.

The effect of the op mutation on the postnataldevelopment of F4/80+ cells(a) F4/80+ cells exhibiting an almost absoluterequirement for CSF-1This group (Table 2a) includes muscle, tendon and dermis inwhich the F4/80+ cell density was normally highest at birth(Table 1), indicating that their requirement for CSF-1 isprenatal. The F4/80+ cells in periosteum, synovial membranes,kidney and the inner nuclear and plexiform layers of the retinawere normally well established at birth and continued toincrease in number during the first 2 weeks of life (Table 1),consistent with a prenatal and early postnatal requirement forCSF-1. Osteoclasts, likely derived from mononuclearphagocyte precursors (Suda et al., 1992; Hofstetter et al.,1992), and similarly dependent on CSF-1, also normallydevelop during the prenatal period (Scheven et al., 1986).Indeed, in contrast to earlier statements (Yoshida et al., 1990;Naito et al., 1991), in op/op mice osteoclasts also fail todevelop during the prenatal period, since the principal trait ofthe op mutation, the osteopetrosis, characterized by the absenceof a distinct medullary cavity, is recognizable both radiologi-cally at birth (Felix et al., 1990a) and histologically at day-17of post-conceptional age in femurs and tibias which normallyat this age are already invaded by marrow (M. G. C. and T.Morohashi, unpublished observations). Taken together, theseobservations indicate that significant expression of the opmutation occurs prenatally.

(b) F4/80+ cells exhibiting partial dependence on CSF-1throughout the postnatal periodA first subgroup (Table 2b), adrenals, bladder, salivary glandsand bone marrow macrophages, with the exception of bonemarrow macrophages, normally appeared postnatally and thustheir partial requirement for CSF-1 is exclusively postnatal. Inthe case of bone marrow macrophages, the partial requirementfor CSF-1 is extended to both pre- and post-natal periods. Asecond subgroup, liver, stomach, gut and spleen (Table 2c),were initially independent of CSF-1, but by two weeks of agedemonstrated a CSF-1 requirement. Since there was no majoreffect of the absence of CSF-1 at birth on these cells, they musteither be independent of CSF-1 or regulated by maternal CSF-1 during the prenatal period (see below). However, all of the


Table 4. Effect of subcutaneous injection of different dosesof CSF-1 on selected F4/80+ cell populations in op/op mice

CSF-1 dose (Units/injection)

Tissue 0 3×104 1×105* 1×105 3×105 1×106

Liver 45** 120 154 177 275 349Spleen 58 64 67 76 87 91Kidney 21 110 312 382 508 1040

op/op mice were injected daily from 3 days of age with CSF-1 or vehiclefor 3 months.

*CSF-1 injected once every 2 days.**Percentage of the F4/80+ cell density of littermate control (+/+ or +/op)

mice.


populations in this subgroup require CSF-1 for their postnataldevelopment and maintenance. At two weeks of age, thespleen, normally at this time a major site of hematopoiesis,exhibits a normal density of F4/80+ cells in op/op mice.However, at this age, we have almost invariably observed asplenomegaly accompanied by a concomitant increase of lessdendritic, more rounded F4/80+ cells, consistent with thereported accumulation of macrophage progenitors (Begg et al.,1993) and probably secondary to the failure of monocytedifferentiation in and migration from the spleen.

A more detailed analysis of the F4/80+ cells exhibiting anabsolute or partial requirement for CSF-1, reveals that someF4/80+ cell populations exhibit delayed development in op/opmice, either partially, as in the case of bone marrow andperiosteal macrophages, or completely, as in the case of themacrophages of the retina. Concomitant with the increase indensity of F4/80+ bone marrow macrophages in op/op mice,there is also an improvement of the osteopetrotic status, asindicated by an increase in the bone marrow area. Indeed, ithas been shown that in older op/op mice (Begg et al., 1993),bone marrow cellularity returns to normal levels by 22 weeksof age and by 35 weeks, the frequency and total number ofF4/80+ cells are normal. These changes in the bone marroware accompanied by an increase in splenic granulocytopoiesisand megakaryocytopoiesis. This improvement of F4/80+ celldensity is restricted to the hematopoietic organs active at theseages (spleen and bone marrow). In fact, in the liver there is nodetectable improvement even at 40 weeks of age (M. G. D., S.M. and E. R. S., unpublished observations). An improvementof the bone remodelling at 45 days of age has been recentlydemonstrated (Wink et al., 1991). The mechanism leading tothe spontaneous remission in these tissues is unknown. Irre-spective of the mechanism involved, the maximum effect ofthe absence of CSF-1 in op/op mice is best studied within thefirst 4-6 weeks of age. The interpretation of morphological andfunctional studies, performed at later time points, especiallythose involving the hematopoietic organs (Naito et al., 1991;Wiktor-Jedrzejczak et al., 1992a; Wiktor-Jedrzejczak et al.,1992b), may have been influenced by this compensatory phe-nomenon.

(c) F4/80+ cell populations virtually independent of CSF-1Some of these F4/80+ cell populations normally developedprenatally and postnatally (Table 2d), while others developedexclusively postnatally (Table 1). F4/80+ cells of lymph nodeand bone marrow monocytes developed postnatally and areclearly completely independent of CSF-1 while Langerhanscells and cells of the thymus could be supported prenatally bymaternal CSF-1, which has recently been shown to cross theplacenta (P. Roth and E. R. S., unpublished observations).However, their substantially normal postnatal developmentand maintenance in the op/op mouse reported here andelsewhere (Takahashi et al., 1992, 1993; Witmer-Pack et al.,1993) is consistent with their total independence of CSF-1.Indeed, the in vitro requirement of granulocyte-macrophageCSF for Langerhans cell viability and function (Witmer-Packet al., 1987), together with the in vivo stimulation of Langer-hans cell recruitment by local injection of this growth factor(Kaplan et al., 1992), confirms that this population is regulatedby factors other than CSF-1. Interestingly, bone marrow

monocytes were the only F4/80+ population to exhibit abovenormal cell densities in op/op mice. Their accumulation in themarrow of 2-week-old op/op mice (Table 2d), occurs at thetime of maximum development of this population in normalmice (Table 1c) and was temporally correlated with a severedeficiency of bone marrow macrophages. These observationsare consistent with the failure of these monocytes to differen-tiate to macrophages in the absence of CSF-1.

Effect of postnatal restoration of circulating CSF-1in op/op miceTissue F4/80+ cells in op/op mice showed three distinctpatterns of response to postnatal restoration of circulating CSF-1 (Table 3).

(a) F4/80+ cells highly responsive to circulating CSF-1In the case of liver and spleen, in which the F4/80+ celldensities were approximately normal in op/op mice at birth anddecreased with increasing age, CSF-1 administration preventedthe postnatal decline. The fact that CSF-1 can cross theplacenta and that the op/op mice used in this study wereoffspring of +/op mothers, strongly suggests that their F4/80+cell densities are normal at birth because in these two organsthey are prenatally regulated by maternal CSF-1. These resultsconfirm an earlier study (Hume et al., 1988), which indicatedthat tissue macrophages of liver and spleen were two majortarget cell populations affected by in vivo administration ofrecombinant human CSF-1 in normal mice. Kidney F4/80+cells, which pre- and postnatally are markedly dependent onCSF-1, exhibited a dramatic response to injected CSF-1, con-sistent with their regulation by the circulating growth factor.The reasons for the failure of circulating maternal CSF-1 tosupport F4/80+ cells in the fetal kidney compared with fetalliver (P. Roth and E. R. S., unpublished observations) are atpresent unknown.

In the op/op spleen, CSF-1 administration completely recon-stituted the MOMA-1+ marginal metallophilic macrophageswith exactly the same morphology, location and pattern seenin normal spleen. Similar specific histological localization afterrestoration of circulating CSF-1 is also observed for the F4/80+cells in the inner nuclear and plexiform layers of the retina andfor osteoclasts (Felix et al., 1990a). As it seems unlikely thatsuch cells have selective access to circulating CSF-1, theirdevelopment is probably dependent on both circulating CSF-1and a cytokine that is locally synthesized or presented atspecific anatomical sites. The function of the splenic marginalmetallophilic macrophages is not clear. It has been suggestedthat they are involved in antigen presentation (Kraal and Janse,1986), the detoxification of endotoxins (Eikelenbloom, 1978)and the direction of lymphocyte traffic in the spleen (Brelinskaand Pilgrim, 1982).

(b) Tissue in which F4/80+ cell densities were partiallyrestored by CSF-1 injectionIn dermis, bladder, salivary glands, stomach and gut, theF4/80+ cells were associated with the epithelia. With theexception of the salivary glands, restoration was limited tothose regions where this association was weak, e.g. in the deepdermal regions and in the lamina propria at the base of themucosa. Interestingly, this is the same region where these cells,which may have been recruited prenatally by maternal CSF-1,

1370

are found at birth. The failure of the epithelially associatedF4/80+ cells to develop in CSF-1 injected op/op mice (e.g. Fig.4E), strongly suggests that their postnatal development isregulated by local presentation of CSF-1. Bone marrowmacrophages failed to be completely restored by CSF-1 admin-istration, suggesting that locally presented CSF-1 is alsonecessary for their complete restoration. In contrast to the otherF4/80+ cells of this group, they developed strongly during thepostnatal period even without administration of CSF-1 (above,Table 3) (Begg et al., 1993). Importantly, both in the case ofspontaneous or induced recovery, these stromal macrophagesare poorly reconstituted in the region of the primary spongiosaand this further suggests that local presentation of CSF-1 at thisparticular region is critical. The recent observation that the pro-teoglycan form of CSF-1 binds bone-derived collagens and isextractable from bone matrix (Ohtsuki et al., 1993) suggeststhat the glycosaminoglycan moiety may mediate selectivelocalization of CSF-1, as originally hypothesized (Price et al.,1992).

(c) Tissues in which F4/80+ densities were unaffectedby CSF-1 injectionA subgroup of macrophages colonizing the dense connectivetissues (muscle, tendon, dermis, periosteum and synovialmembrane), which did not benefit from transplacental CSF-1and failed to develop during the prenatal period, was also unre-sponsive to injected CSF-1. These tissue macrophages are mostlikely to be dependent on locally presented CSF-1 (see above).

The densities of F4/80+ macrophages in the thymus, lymphnode, epidermis (Langerhans cells) and of the bone marrowmonocytes were not decreased in op/op mice and were notaffected by CSF-1 injection. This observation, together withthe relatively normal early development of splenic F40/80+macrophages are in agreement with recent immunologicalstudies in which it has been shown that op/op mice possessnormal in vivo phagocytic function, normal delayed typehypersensitivity and normal humoral and cellular immuneresponses to sheep red blood cells (Wiktor-Jedrzejczak et al.,1992a).

CSF-1 has been shown to stimulate the spreading of culturedmacrophages (Boocock et al., 1989) and there are distinct dif-ferences in the morphology of macrophages grown in thepresence of CSF-1 and those grown in the presence of granu-locyte-macrophage CSF (GM-CSF), which are more rounded(Falk and Vogel, 1988; Akagawa et al., 1988). Previous studieshave reported that uterine, splenic and liver macrophages fromop/op mice (Naito et al., 1991; Pollard et al., 1991) are moreround and possess less developed organelles than their coun-terparts in normal mice. In the present study a smaller, morerounded appearance of F4/80+ cells in op/op mice comparedwith littermate control mice was also noted in the spleen, liver,kidney, adrenal gland, bladder, salivary glands, bone marrowand gastrointestinal tract. In most situations where CSF-1administration elicited a recovery of the F4/80+ populationthere was at least some reversion towards normal macrophagemorphology. In the case of the MOMA-1+ marginal metal-lophilic macrophages and retinal microglia there was completeor even exaggerated reversion. The functional significance ofthe more rounded morphology of the CSF-1-dependentmacrophages in op/op mice is not clear but is possibly relatedto their failure to differentiate terminally in the absence of

CSF-1. Interestingly, there was no detectable difference in themorphology of F4/80+ cell populations whose densities wereunchanged in op/op mice.

The approach used here to analyze the requirement of tissuemononuclear phagocyte populations for local and humoralCSF-1 has several limitations. Neither the mouse glycoproteinor proteoglycan forms of CSF-1, which naturally occur in thecirculation, were used because human recombinant CSF-1 wasthe only form of CSF-1 available in sufficient quantities. Thismay mean that in some situations where lack of regulation bycirculating recombinant CSF-1 occurred, local regulation bydifferential localization of circulating glycosylated CSF-1might actually occur. Secondly, because of the difficulty indetermining what dose of CSF-1 to inject, we chose to usehigher concentrations than normal in order to ensure thatexcess rather than limiting concentrations of circulating CSF-1 would be available. This has helped our interpretation of thedata in situations where F4/80+ populations were unaffectedsince they were unaffected at very high concentrations.However, since the doses used were significantly higher thanthose required for regulation of the responding populations(Table 4), it is possible that some populations that werepartially or completely corrected are in fact not normallyregulated by circulating CSF-1. An additional limitation isassociated with the difficulties of studying the fetal op/opF4/80+ populations from fetuses carried by op/op mothers.Because of the very poor fertility of op/op×op/op mating pairs(Pollard et al., 1991), this was not possible in the present study.

A simple interpretation of the data is presented in summaryform Table 5, where tissue macrophages are groupeddepending on the nature of their requirement for CSF-1 andwhether it is required prenatally and/or postnatally. In general,


Table 5. Summary of the role of CSF-1 in the regulation oftissue macrophage development

CriterionCSF-1 requirement

postnatal for Timing circulating

Tissue detection Degree prenatal postnatal CSF-1

Muscle F4/80 complete + − noneTendon ,, ,, + − ,,Dermis ,, ,, + − partial/none**Periosteum ,, ,, + +(early) noneSynovium ,, ,, + +(early) ,,Kidney ,, ,, + +(early) completeRetina ,, ,, + +(early) ,,Adrenals ,, partial − + noneBladder ,, ,, − + partial/none**Sal. gland ,, ,, − + partialBm macr ,, ,, + + partialLiver ,, ,, +† + completeStomach ,, ,,* − + partial/none**Gut ,, ,,* − + partial/none**Spleen ,, ,, +† + completeEpidermis ,, none ? − noneThymus ,, ,, ? − ,,Lymph node ,, ,, − − ,,Bm mono ,, ,, − − ,,

Spleen MOMA-1 complete − + complete

*Localized sub-populations within these tissues are completely dependent.**Localized sub-populations exhibited different responses to restoration of

circulating CSF-1 (see text).†Regulated prenatally by circulating maternal CSF-1 (P. Roth, M.G.D. and

E.R.S., unpublished observations).

Response to


macrophages requiring CSF-1 for their establishment or main-tenance appear to have trophic or scavenger roles important inorganogenesis and tissue remodelling (physiologicalprocesses), whereas CSF-1-independent macrophages areprimarily involved in immune and inflammatory responses(pathological processes). Further exploration of the functionsof the CSF-1-dependent macrophages, utilizing the op/opmouse, is warranted in view of the aspects of the op phenotype,such as reduced weight, poor fertility, hypoplastic andhypotrophic dermis and synovial membrane and neuromuscu-lar disfunction that might result from their absence.

We thank F.-C. Chuan, J. Portenier, R. Rubli, U. Mausli and R.Zadeh for excellent technical assistance and Dr Phillip Roth for crit-ically reviewing the manuscript. This work was supported by SwissNational Science Foundation grants 3.894.0.88 and 32.31272.91., byAmerican Cancer Society Grant DB-28 and a Monique Weill-CaulierAward (to J. W. P.), by NIH grant CA 32551 and a grant from theLucille P. Markey Charitable Trust (to E. R. S,) and the AlbertEinstein Core Cancer Grant P30-CA 13330.

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(Accepted 18 February 1994)


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