Ontogenic lapositive

development of macrophage subpopulations and dendritic cells in fetal and neonatal rat spleen

Motohiro Second

Takeya

Department

and of

Kiyoshi

Pathology,

Takahashi

Kumamoto

University

School

Abstract: Development, differentiation, and distribution of macrophage subpopulations and Ia dendritic cells in the fetal and neonatal rat spleen were investigated by means of double immunohistochemical staining and immunoelectron microscopy. To characterize these cell populations, a panel of anti-rat macrophage monoclonal antibodies (RM-1, ED2, ED3, TRPM-3, Ki-M2R) and an anti-rat Ia antibody (0X6) were used. In the fetal rat spleen, macrophages were first detected by RM-1 at fetal day 15. ED2 and/or Ki-M2R macrophages appeared at fetal day 16. TRPM-3 and/or ED3 macrophages ap-

The phages humans

day 15. Ia cells

In the fetal and neonatal gradually increased, their

period, shape

dendritic, and they matured into interdigitating the inner periarteriolar lymphatic sheath. In onIa dendritic cells were not stained with ED2 or TRPM-3. These results suggest that ED2 macrophages, TRPM-3 macrophages, and Ia dendritic cells are distinct cell lines that pursue independent developmental processes in spleen ontogeny. J. Leukoc. Biol. 52: 516-523; 1992. Words:

double

immunohistochemical

monoclonal

antibodies staining

.

.

Wistar

microscopy

macroand in and ne-

AND

METHODS

white

rats

maintained

under

routine

laboratory

con-

Antibodies Mouse anti-rat macrophage [101 and RM-i [11] were M2R was kindly provided Pathology, Kiel University

Macrophages form a heterogeneous cell population with regard to their functions, phenotypic expressions, and tissue localizations. The different organs contain specially differentiated organ-specific macrophages. Because the spleen has a unique morphological feature with distinct compartments, different macrophage populations reside in different compartments [1]. Most of the macrophages are located in the cord of Billroth in the red pulp (red pulp macrophages). The other macrophages reside in white pulp, which consists of three major compartments: the periarteriolar lymphatic sheath (PALS), the lymphoid follicles, and the marginal zone

of Leukocyte

Biology

Volume

52,

November

monoclonal antibodies TRPM-3 produced in our laboratory. Kiby Dr. H-H. Wacker, Institute of [12]. ED2 and ED3 [9] were pur-

chased from Serotec (Oxford, UK). Monoclonal mouse anti-rat Ia (OX6, Sera Lab, Sussex, UK) and polyclonal rabbit anti-rat immunoglobulin M (IgM)() antibody ( MBL, Nagoya, Japan) were also used. For immunohistochemistry, RM-i, TRPM-3, and Ki-M2R were purified with a protein A-Sepharose affinity column and used at a di-

[2]. Marginal metallophilic macrophages are located in the periphery of PALS at its border with the marginal zone. Marginal zone macrophages are located in the marginal zone itself. Tingible body macrophages are observed in the germinal center of the follicles. In addition, the cells with dendritic morphology called interdigitating cells are seen in the inner PALS [3].

Journal

and [4-6] fetal

ditions with free access to food and water were used. The first gestational day (fetal day 0) was decided by the presence of a vaginal plug in the morning after mating. Developing spleens were obtained from rat fetuses at fetal days 14 to 21, from neonates at 0 to 7 days, and from young rats at 10, 14, 17, 21, 24, 28, 35, 42, and 49 days after birth. At least three rat fetuses, newborns, or young adults were examined at each time point.

INTRODUCTION

516

lymphocytes in animals about the

Animals

immunohistochemistry

immunoelectron

Japan

development of splenic has been studied extensively [7, 8]. However, information

MATERIALS

became cells in togeny,

Key

Kumamoto,

onatal development of splenic macrophages is not sufficient, partly because of the lack of markers for discriminating different macrophage subpopulations. The production of specific monoclonal antibodies against different macrophage populations has provided an important tool for studying their heterogeneity [9-12]. Using a panel of antimacrophage antibodies, several investigators have studied the ontogenic development of macrophages in various lymphoid and nonlymphoid organs [13-22]. However, the development of macrophage subpopulations in the fetal and neonatal spleen is still not fully understood. In the study reported here, we examined the ontogenic development of macrophage subpopulations in the fetal and neonatal rat spleen using monoclonal antibodies specific for them.

peared a day later. During the fetal and neonatal development, ED2 and TRPM-3 macrophages differentiated independently, maturing into red pulp macrophages and marginal metallophilic and marginal zone macrophages respectively. Intimate topographical relations were observed between ED2 macrophages and hematopoietic cells and between TRPM-3 macrophages and marginal zone lymphocytes. Ia cells were first observed around arterioles at fetal the number of

of Medicine,

Abbreviations: APAAP, alkaline phosphatase anti-alkaline phosphatase; DAB, 3,3’-diaminobenzidine; 1gM, immunoglobulin M; PALS, periarteriolar lymphatic sheath; PBS, phosphate-buffered saline; PLP, periodatelysine-paraformaldehyde. Reprint requests: Motohiro Takeya, Second Department of Pathology, Kumamoto University School of Medicine, 2-2-I Honjo, Kumamoto 860, Japan. Received March 9, 1992; accepted July 1, 1992.

1992

lution of 1:200. ED2, ED3, fluid diluted 1:100. Anti-IgM(j) tion of 1:200.

and

OX6 antibody

were was

used used

as ascites at a dilu-

Selected

Immunohistochemistry after resection, the splenic tissues were fixed in periodate-lysine-paraformaldehyde (PLP) fixative for 4 h at 4#{176}C.After 4-h washes with phosphate-buffered saline ( PBS) containing 10, 15, or 20% sucrose and 20% sucrose + 5% glycerol, they were embedded in tissue-embedding medium (Miles, Elkhart, IN), frozen in dry ice-acetone, and Immediately 2%

cut into 6-sm-thick sections with a cryostat (Bright, Huntingdon, UK). After inhibition of endogenous peroxidase activity according to the method of Isobe et al. [23], the sections were incubated with each monoclonal or polyclonal antibody for 60 mm at room temperature. After three washes with PBS for 5 mm each, peroxidase-conjugated sheep anti-mouse

immunoglobulin

[F(ab’)2]

diluted

1:100

(Amer-

sham, Amersham, UK) was reacted for 60 mm as the second step. To visualize peroxidase activity, 3,3’-diaminobenzidine (DAB) (Sigma, St. Louis, MO) was used as substrate in 0.05 M Tris-HC1 buffer (pH 7.6) containing 0.01% H202. Nuclear stain was performed with hematoxylin. Control slides were incubated with nonimmunized mouse serum or PBS

instead

of the

primary

antibody;

they

were

invariably

negative.

Double

immunohistochemical

Immunoelectron

staining

the first step, the frozen sections were stained by the indirect immunoperoxidase method mentioned above using one of the monoclonal antibodies. After visualizing this reaction stain brown by DAB, the sections were treated with 0.1 M glycine-HC1 buffer (pH 2.2) for 60 mm with four changes to remove the first and second antibodies reacted. Then the same sections were incubated overnight with one of the other antimacrophage monoclonal antibodies. After three washes with Tris-buffered saline (pH 7.6), the sections were treated by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method using an APAAP kit (DAKO, Carpinteria, CA). To visualize the reaction of APAAP, the sections were stained blue in a substrate made up of0.2 mM naphthol ASMX phosphate (Sigma), 1 mM fast blue BB salt (Sigma), and 1 mM levamisole (Aldrich, Milwaukee, WI) in 50 mM Tris-HC1 buffer (pH 8.5) for 20 mm. To confirm the reaction specificities of each step, the first antibodies in the first or second step were omitted in control experiments. Double staining of rabbit anti-rat IgM() polyclonal antibody and one of the antimacrophage monoclonal antibodies was performed as above without the treatment with glycineHC1 buffer.

As

TABLE

1. of

Reactivity

Antibody

Isotype

Site

RM-1 ED2 Ki-M2R ED3 TRPM-3

IgGl IgG2a IgA IgG2a IgG2a

Cell Cell Cell Cell Cell

membrane membrane membrane membrane membrane

0X6

IgGl

Cell

membrane

anti-IgM(gz)

Polyclonal

Cytoplasm,

of Antirat

splenic

0.05 series thin psala,

were

sliced

M cacodylate buffer (pH of ethanols, and embedded sections were cut on an Sweden) and observed

microscope nyl acetate.

(Hitachi,

with

razor

blades

and

Tokyo,

7.4),

dehydrated flat in epoxy Ultrotome Nova in a Hitachi-12A

Japan)

after

staining

in a graded resin. Ultra(LKB, Upelectron with

ura-

RESULTS Localization rat spleen

of macrophage

subpopulations

in the adult

Table 1 summarizes the positive cells of each antibody in the adult rat spleen. The reactivity of each antimacrophage monoclonal antibody in the adult spleen is almost the same as those described in the previous reports [9-12]. RM-1 was reactive with most splenic macrophages and interdigitating cells (data not shown). ED2, Ki-M2R, ED3, and TRPM-3 recognized different macrophage subpopulations. The former two were reactive with red pulp macrophages, whereas the latter two labeled marginal metallophilic macrophages and marginal zone macrophages. Subset specificities of these antibodies were well documented by double immunohistochemical staining. In the double staining of ED2 and TRPM-3 (Fig. la), ED2 recognized red pulp macrophages exclusively, whereas TRPM-3 labeled marginal metallophilic macrophages and marginal zone macrophages. Interdigitating cells in the inner PALS and B cells in the marginal zone and lymphoid follicles were detectable by their expression of Ia antigen (Fig. ib) as reported previously [5, 6, 24]. B lymphocytes in the marginal zone were strongly positive for 1gM, and those in the follicles were moderately positive for 1gM (data not shown). In the immunoelectron microscopic study, ED2 distinctly labeled the cell membrane of red pulp macrophages (Fig. 2a). TRPM-3 clearly labeled the cell membrane of marginal metallophilic macrophages, which formed a rim surrounding the PALS (Fig. 2b).

Development subpopulations At fetal attached

and difterentiation of macrophage in the fetal rat spleen

day 14, the spleen to the dorsal wall

Antibodies

in

Reactive

Adult

membrane

and

Rat

as the red At this stage

streak small

Spleen

most macrophages, interdigitating macrophages macrophages metallophilic macrophages, marginal metallophilic macrophages, marginal

Interdigitating

cells,

B lymphocytes

in

Takahashi

was recognizable of the stomach.

cells

Monocytes, Red pulp Red pulp Marginal Marginal

Takeya

tissues

fixed in 4% PLP at 4#{176}C for 4 h. After washing with PBS, the slices were incubated with TRPM-3 or ED2 overnight at 4#{176}C. Then they were reacted with peroxidase-labeled anti-mouse immunoglobulin [F(ab’)2] for 3 h at room temperature. Peroxidase activity was visualized as described above. Then the tissues were postfixed with 1% osmium tetroxide in

recognition

cell

microscopy

cells

zone zone

macrophages macrophages

B lymphocytes follicles

Development

and

marginal

of rat

splenic

zone

macrophages

517

Fig. 1. Immunohistochemical red pulp. (a) In double staining, and marginal zone macrophages. follicles.

staining of adult rat spleen. A, arteriole; F, lymphatic ED2 (brown) specifically labels red pulp macrophages, x 100. (b) OX6 (anti-Ia) is reactive with interdigitating

populations macrophages However,

(Fig. and TRPM-3 around

were seen farther away (Fig. 3g). ated mainly around the arterioles ally increased. IgM cells were spleen.

Journal

Development subpopulations rat adults

arterioles,

micrographs on the cell

of Leukocyte

whereas

of adult membrane

Biology

ED2

rat spleen. of marginal

Volume

macrophages

(a) Cell membrane of a red metallophilic macrophages

52,

November

Ia

dendritic and their not observed

cells were situnumber graduin the fetal

and differentiation of macrophage in the spleen of rat neonates and young

In the very early neonatal period, from day 0 to 5, many Ia cells with short and blunt cell surface projections accumulated around the arterioles (Fig. 3h). ED2 (and Ki-M2R) macrophages and TRPM-3 (and ED3) macrophages were scattered outside the Ia dendritic cells. In double staining, Ia cells were not reactive with ED2 or TRPM-3 (Fig. 3i). At day 2, IgM B lymphocytes were first observed around the arterioles, and their number increased thereafter to form primitive marginal zone. At day 5, ED2 macrophages were found outside the primitive marginal zone (Fig. 3j), whereas

3g). In contrast to the adult spleen, ED2 TRPM-3 macrophages were intermixed. macrophages tended to accumulate the

Fig. 2. Immunoelectron TRPM-3 are observed

518

sheath; RP, macrophages and lymphoid

xlOO.

clusters of erythroblasts were scattered between mesenchymal cells. At fetal day 15, a few RM-1 macrophages appeared dispersively. Some Ia cells were also seen around the arterioles (Fig. 3a). Although these cells were round or oval, they were thought to be the precursor cells of interdigitating cells, not of B cells, because they were also labeled by RM-1 in double staining (Fig. 3b). At fetal day 16, a few ED2 (Fig. 3c) and Ki-M2R cells appeared. TRPM-3 (Fig. 3d) and ED3 cells first appeared at fetal day 17. Through the fetal stage, RM-P macrophages outnumbered ED2 macrophages (Fig. 3e) and TRPM-3 macrophages (Fig. 30. In double staining, most ED2 macrophages (Fig. 3e) and TRPM-3 macrophages (Fig. 31) were also labeled by RM-1, whereas ED2 and TRPM-3 labeled different macrophage

closely

follicle; MZ, marginal zone; P, periarteriolar lymphatic whereas TRPM-3 (blue) stains marginal metallophilic cells in inner PALS and B cells in the marginal zone

1992

pulp (M).

macrophage Bar, I zm.

(M)

is labeled

by ED2.

(b)

Reaction

products

of

Takeya

and

Takahashi

Development

of rat splenic

macrophages

519

Fig. 3. Immunostaining cells are seen around are labeled by RM-l,

of fetal and neonatal rat spleen. an arteriole at fetal day 15. x520. whereas some RM-l” cells (arrows)

A, arteriole; (b) Double are devoid

MZ, marginal zone; P, periarteriolar staining with Ia (brown) and RM-l of staining with Ia. x800. (c) ED2

lymphatic (blue) at fetal macrophages

sheath; RP, red pulp. (a) A few Ia” day 15. Most La’ cells (arrowheads) (arrowheads) first appeared in fetal

spleen at day 16. x 520. (d) TRPM-3 macrophages (arrowheads) first appeared in fetal spleen at day 17. x 520. (e) Double staining with ED2 (brown) and RM-l (blue) at fetal day 17. Arrowheads indicate ED2 and RM-l” macrophages. x400. (I) Double staining with TRPM-3 (brown) and RM-l (blue) at fetal day 18. Arrowheads indicate TRPM-3” and RM-l” macrophages. x400. (g) In double staining of a fetal spleen at day 20, ED2 (brown) and TRPM-3 (blue) recognize different macrophage populations. x 260. (h) Ia” cells are mainly observed around an arteriole at I day after birth. x 260. (i) Double staining of a neonatal spleen at 1 day after birth. TRPM-3” cells (brown) are scattered outside the Ia” cells (blue). x 400. (j) At neonatal day 5, ED2 (blue) macrophages were found outside the primitive marginal zone, which is formed by 1gM” B lymphocytes (brown). x200. (k) ED2 (brown) macrophages are seen outside the rim of TRPM-3” marginal metallophilic macrophages (blue) at neonatal day 5. x 260.

TRPM-3 marginal metallophilic macrophages formed a rim surrounding the primitive PALS, although the cells were not continuously arrayed (Fig. 3k). On immunoelectron microscopy of early neonatal spleen, TRPM 3 macrophages extended cytoplasmic processes among the surrounding lymphocytes (Fig. 4a). ED2 macrophages, on the other hand, were usually found between the hematopoietic cells (Fig. 4b). After day 5, the PALS gradu-

loped, ED2 and TRPM-3 labeled different macrophage subpopulations. Thus, it is considered that ED2 and TRPM-3 macrophage subpopulations differentiate independently through the fetal and neonatal life and mature into red pulp macrophages and marginal metallophilic and marginal zone macrophages, respectively. The difference between these two major macrophage subpopulations was also observed in an experiment on liposome-mediated elimination of splenic macrophages by van Rooijen et al. [26], who reported that red pulp macrophages and marginal zone macrophages showed a striking difference in the kinetics of their reappearance. Considering their result, our data indicate that these

ally expanded. At day 10, TRPM-3 marginal metallophilic macrophages formed a complete rim between the PALS and the marginal zone, and a few TRPM-3 marginal zone macrophages appeared among IgM marginal zone B lymphocytes (Fig. Sa). The marginal zone bridging channels [25] were also recognized at this stage (Fig. 5a). At day 14, IgM and Ia B lymphocytes accumulated inside the rim of TRPM-3 marginal metallophilic macrophages to form primary lymphoid follicles. At the same day, many Ia interdigitating cells with elongated cell processes were observed in the inner PALS (Fig. Sb). At day 28, germinal centers developed in the lymphoid follicles as reported previously [4]. Thus the architecture of the white pulp became very similar to that of the adult spleen. Figure 6 schematically summarizes the development ofmacrophage subpopulations and Ia dendritic cells in the rat spleen.

two

major

splenic

macrophages

are

distinct

cell

lineages.

DISCUSSION Immunohistochemistry with monoclonal antibodies readily permits the differentiation of various cell types. In recent years, many antimacrophage monoclonal antibodies have been used to investigate macrophage heterogeneity. Using a number of monoclonal antibodies, Buckley et al. [1] demonstrated in the human spleen that different phenotypic subsets of macrophages are present in discrete microanatomic locations. Among the antirat monoclonal antibodies used in this study, RM-1 is considered to be an anti-pan-macrophage monoclonal antibody that recognizes almost all macrophages, monocytes, and dendritic cells such as interdigitat ing cells or Langerhans cells [11]. ED2 and Ki-M2R recognize resident macrophages in various organs but not blood monocytes. In the spleen, they react exclusively with red pulp macrophages [9, 12]. TRPM-3 rophage subpopulations, such macrophages and marginal zone sinus macrophages in the lymph rophages, but not blood monocytes reaction pattern similar to that ED2 and membrane

TRPM-3 among

these two antibodies tron microscopic It is noteworthy express TRPM-3 at the fetal period,

520

Journal

recognizes certain macas marginal metallophilic macrophages in the spleen, nodes, and omentum mac[10]. ED3 revealed a of TRPM-3 [9]. Because

gave a clear reaction these macrophage-specific were study. that the and ED2 when

of Leukocyte

mainly

used

to the for

the

cytoplasmic antibodies, immunoelec-

different macrophage populations through their development. Even no splenic compartment is deve-

Biology

Volume

52,

November

Fig.

Immunoelectron micrograph of neonatal spleen at day 3. (a) A macrophage (M) extends its cytoplasmic processes between surrounding lymphocytes. Distinct reaction products (arrowheads) are seen on the whole plasma membrane of the macrophage. (b) An ED2 macrophage (M) shows intimate cell-to-cell contact with surrounding erythroblasts. Arrowheads indicate reaction products of ED2. Bar, I gzm. TRPM-3”

1992

4.

Fig.

5. Immunostaining

of young rat spleen. A, arteriole; BC, marginal zone bridging channel; F, lymphoid follicle; MZ, marginal zone; P periarteriolar RP, red pulp. (a) Double staining with 1gM (brown) and TRPM-3 (blue) at day 10. TRPM-3 marginal metallophilic macrophages (blue) form a complete rim between PALS and marginal zone. A marginal zone bridging channel is also seen. x 130. (b) Ia dendritic cells are seen in the inner PALS. Lymphocytes in marginal zone and a primary lymphoid follicle are also positive for Ia. x 100. lymphatic

The in the

sheath;

marginal rat [27]

zone is the largest white and has a unique property

pulp compartment both morphologi-

cally and functionally. Marginal zone B cells are considered to be a distinct cell lineage and are easily detectable by their expression of IgM() [28]. Marginal zone macrophages play a special role in presenting thymus-independent antigens to surrounding B lymphocytes [29]. In the present study, TRPM-3 macrophages were already observed around the

participate of marginal

in the zone

:4f ts;s5;::c

ladendritic

N

lOd

cell macrophage

28d

14d 6. Schematic BC,

RP,

red

marginal

drawing zone

differentiation

ED2 macrophage

TRPM4’

Fig.

and

)RP

5d

20 d

--

ole;

emigration B cells.

* ):::

fetal

dered to processes

4’

A#{149}PA

V#{174}

arterioles in the fetal period before the emigration of IgM marginal zone B cells. After the appearance of marginal zone B cells shortly after birth, an intimate topographical relation between TRPM-3 macrophages and marginal zone B cells was observed by immunoelectron microscopy. Taking these findings together, TRPM-3 macrophages are consi-

of the ontogenic bridging

channel;

development PF, primary

of ED2 lymphoid

and TRPM-3” follicle; GC,

macrophage germinal

subpopulations and center; MZ, marginal

Ia dendritic cells in rat spleen. A, arterizone; P, periarteriolar lymphatic sheath;

pulp.

Takeya

and

Takahashz

Development

of rat splenic

macrophages

521

monoclonal

In rodents such as rats and mice, the spleen, particularly the red pulp, is a major site of hematopoietic activity in the fetal and neonatal life, and splenic hematopoiesis persists until adulthood [30]. As ED2 macrophages were observed at the center of the erythroblastic islands, these cells may play an important role in fetal and neonatal erythropoiesis. Considering the different microanatomical localizations of ED2 and TRPM-3 macrophages, it is of great interest to note that these two macrophage subpopulations share special microenvironments related to their functions at the early ne-

10.

11.

of interdigitating the fetal and

cells neonatal

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Wacker,

Leukoc.

( 1987)

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