ORIGINAL ARTICLE

B-1 cells are pivotal for in vivo inflammatory giant cell formation

Cristina Stewart Bittencourt Bogsan, Ronni Romulo Novaes e Brito, Maiko da Cruz Palos, Renato

Arruda Mortara, Sandro Rogerio Almeida, Jose Daniel Lopes and Mario Mariano

Department of Microbiology, Immunology and Parasitology, Federal University of Sao Paulo, Sao Paulo, Brazil

Received for publication:

15 October 2003

Accepted for publication:

18 March 2005

Correspondence:

Prof Mario Mariano

Rua Botucatu 862

4th floor, 04023-9000 Sao Paulo

Brazil

Tel.: +55 11 5496073

Fax: +55 11 55723328

E-mail: mariomu@uol.com.br

Abstract

The mechanisms that govern giant cell (GC) formation in inflammatory, neoplastic

and physiologic conditions are far from being understood. Here, we demonstrate that

B-1 cells are essential for foreign-body GC formation in the mouse. GCs were analysed

on the surface of glass cover slips implanted into the subcutaneous tissue of the

animals. It was demonstrated that GCs are almost absent on cover slips implanted

into the subcutaneous tissue of BALB/c or CBA/N X-linked immunodeficient mice. As

these animals do not have B-1 cells in the peritoneal cavity, they were reconstituted

with B-1 cells obtained from cultures of adherent mouse peritoneal cells. Results

showed that in B-1-reconstituted animals, the number of GCs on the implant surface

surpassed the values obtained with preparations from wild animals. In animals select-

ively irradiated (pleural and peritoneal cavities) to deplete these cavities of B-1 cells,

GCs were also not formed. Enriched suspensions of B-1 cells grown in culture were

labelled with [3H]-tymidine and injected into the peritoneal cavity of naive mice before

implantation of glass cover slips. After 4 days, about 17% of mononuclear cells had

their nuclei labelled, and almost 70% of GCs had one or more of their nuclei labelled

when analysed by histoautoradiographic technique. A few GCs expressed an immuno-

globulin M when analysed by immunostaining and confocal microscopy. Overall, these

data demonstrate that B-1 cells are pivotal in the mechanisms of foreign-body GC

formation in the mouse.

Keywords

B-1 cells, inflammatory giant cell, macrophages

Although scientific reports found in the older literature

describing multinucleated cells are contemporaneous with

the emerging concept of Virchow’s cellular theory, the origin,

properties and fate of giant cells (GCs) are still poorly under-

stood. As reviewed by Harris (1970), the first description of

cell multinucleation was in 1838 with Muller who first

observed such cells in tumours of vertebrates. Later, in

1855, Rokitansky referred their presence in tuberculous tis-

sue. In 1858, Robin reported their presence among bone

marrow cells, and Virchow and Langhans showed that they

were present in a variety of normal, inflammatory and neo-

plastic lesions in 1858 and 1868, respectively. Although at

that times the biological significance of multinucleation was

unexplained, Metchnikoff (1968) hypothesized that fusion of

phagocitic cells to form ‘plasmodia’ was a characteristic cel-

lular defence mechanism and suggested, on a mechanistic

basis, that fusion of macrophages would result in a more

efficient cellular element to cope with parasites. Although

Int. J. Exp. Path. (2005), 86, 257–265

� 2005 Blackwell Publishing Ltd 257

provocative, his hypothesis has not yet been fully confirmed

or refuted. However, GCs have the capacity to phagocytose

particles via CR1 and Fc receptors (Mariano & Spector 1974;

Papadimitriou & Walters 1979). This phagocytic capacity

was less effective when compared with that of inflammatory

macrophages (Papadimitriou et al. 1975), but it remains to be

clarified whether or not these cells are endowed with bacter-

icidal ability.

GCs are a hallmark histological feature of some types of

chronic inflammation and neoplasia. They occur in various

conditions where the pathological agent is known, e.g.

bacterial, viral, parasitic and fungal infections as well as

where it is not known, e.g. sarcoidosis and rheumatoid

arthritis. GCs are formed in foreign-body lesions induced

by asbestos, carbon, silica and sutures (Papadimitriou &

Walters 1979; Chambers & Spector 1982; Anderson 2000).

They are observed at the tissue–material interface of med-

ical devices implanted in soft and hard tissue and remain

for the lifetime of the device implantation (Anderson 2000).

Foreign-body implantation in man (Keymeulen &

Dillemans 2004) and animals (Brand et al. 1977) can

induce the development of sarcomas. It was also demon-

strated that GCs can undergo aberrant mitosis which

results in chromosome pulverization and micro and poly-

ploid nuclei formation (Mariano & Spector 1974). The

possible relationship between these two phenomena has

not been addressed in the literature.

About 30 years ago, experimental evidences suggested, as

Metchnikoff predicted, that GCs or polykaria are formed in

foreign-body lesions induced in mice by fusion of inflamma-

tory mononuclear cells (Gillman & Wright 1966; Mariano &

Spector 1974). However, since then there has been little

advance in knowledge about the mechanism of the fusion

process itself. It is not clear how mononuclear cell fusion is

induced in vivo, and whether different mechanisms are

involved in different pathological conditions.

A number of possible mechanisms have been discussed in

the literature about how the formation of multinucleated

GCs might be induced in vitro and in vivo. In vitro, these

include the use of conditioned medium (Postlethwaite et al.

1982; Kreipe et al. 1988; Most et al. 1990), several different

cytokines (Nagasawa et al. 1987; Weinberg et al. 1987;

McInnes & Rennick 1988; Enelow et al. 1992a, 1992b;

Fais et al. 1994, 1997; Kazazi et al. 1994; McNally &

Anderson 1995), the addition of lectins alone or in combin-

ation with interferon-g (Chambers 1978; Takashima et al.

1993) and the addition of anti-macrophage antibodies or

phorbol myristate acetate or a combination thereof

(Chambers 1978; McInnes & Rennick 1988; Hassan et al.

1989). Despite all these efforts to understand the

mechanisms of multinucleation, it is not known why GCs

are spontaneously formed during long-term culture of differ-

ent cell types (Most et al. 1997).

Few studies have addressed the question of whether in vivo

multinucleate GCs originate from fusion of macrophages or

other cell types (Gillman & Wright 1966; Spector & Lykke

1966; Mariano & Spector 1974; Chambers 1977; Most et al.

1997). Some time ago, Mariano & Spector (1974) showed

experimental evidence that these cells are formed in foreign-

body lesions by fusion of two cell types called by the authors

old and young or newly arrived ‘macrophages’. Based on the

demonstration that structural chromosome alterations occur

in inflammatory cells in the cover slip model, these authors

hypothesized that the ‘newly arrived cells’, by recognizing

genetically altered cells, would result in cell fusion with

consequent GC formation. Chambers (1977) argued this

hypothesis further with evidence that GCs are formed when

more than one macrophage tempts to phagocytose a single

particle resulting in cell membrane fusion. Nevertheless,

neither the nature of the two cell populations involved in

GC formation nor the concomitant phagocytosis hypothesis

proposed by Chambers (1977) have been fully validated or

refuted.

We have recently demonstrated that B-1b cells – a subtype

of B cell lineage which express surface markers such as

Mac-1, IgM, IgD and B220 promiscuously (Iacomini &

Imanishi-Kari 1992) and found predominantly in pleural

and peritoneal cavities – have a peculiar morphology

(Abrahao et al. 2003), down-regulate macrophage functions

in vitro via IL-10 (Popi et al. 2004) and proliferate in long-

term cultures of adherent mouse peritoneal cells. Further,

these cells can be modulated towards a novel type of mono-

nuclear phagocyte as demonstrated by Borrello et al. (2001),

secrete IgM in vitro and can migrate from the peritoneal

cavity to a non-specific inflammatory lesion induced in

mice (Almeida et al. 2001). Based on these observations

and using the X-linked immunodeficient (Xid) mice, a

mouse strain that have Bruton’s tyrosine kinase mutation

and lacks B-1 cells (Scher 1982; Hardy et al. 1983), we

investigated whether B-1 cells might be involved in foreign-

body GC formation. Following this hypothesis, here we

present evidences that B-1 cells do indeed participate in GC

formation in a foreign-body model of inflammation.

Materials and methods

Animals

BALB/c, BALB/c Xid, CBA/J and CBA/N Xid mice, 8–12-

week-old, were used. Mice were obtained from animal

258 C. S. B. Bogsan et al

� 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 257–265

facilities of the Department of Immunology, University of Sao

Paulo and UNICAMP, Sao Paulo, Brazil.

B1-b-cell cultures

B1-b cells were produced according to the method proposed

by Almeida et al. (2001). Briefly, the peritoneal cavities of

normal BALB/c mice were washed with 6 ml of RPMI 1640

medium (Sigma Chemical Co., St Louis, MO, USA). Cells

were cultured at 37 �C in a humidified atmosphere containing

5% CO2 in RPMI 1640 medium supplemented with 10% of

foetal calf serum (FCS) in 75 cm2 culture vessels (BD Falcon,

Bedford, MA, USA). After 1 h, the supernatant of these cul-

tures were discarded, and the adherent cells were harvested

and re-suspended in RPMI 1640 medium supplemented with

10% FCS. After 7 days of culture without changing the med-

ium, about 90% of the cells in the supernatant of the cultures

were characterized as B1-b cells by flow-cytometry analysis.

The cover slip model

Round glass cover slips (13 mm) were implanted into the

subcutaneous tissue of mice from different experimental

groups as described (Ryan & Spector 1970; Mariano &

Spector 1974). After 4 days of implantation, cover slips were

removed and the cells on their surface fixed in 3.5% formalin

for 1 h, stained with haematoxilin-eosin or used for histoau-

dioradiographic analysis. Using a 20x objective, the mean of

the total number of multinucleated cells/microscopic field

obtained from 10 microscopic fields per cover slip was

recorded. The number of nuclei per GC was also evaluated.

A minimum of five animals with two implanted cover slips in

each was used to constitute the experimental groups.

Confocal microscopy

Cover slips were implanted in normal BALB/c mice, removed

after 4 days and the cells on their surface prepared for the

detection of immunoglobulin M (IgM) and F4/80 (Santa Cruz

Biotechnology, Santa Cruz, CA, USA) using immunostaining

and confocal microscopy as described previously (Abrahao

et al. 2003).

Flow cytometry

Two-colour flow cytometry was used to determine the pheno-

type of peritoneal cells collected from normal, irradiated and

reconstituted mice. Cells were collected by peritoneal lavage

with phosphate-buffered saline (PBS). After centrifugation,

cells were washed with PBS plus 10% FCS, and 10 000 events

were stained with monoclonal antibodies (mAbs) directed to

mouse CD45/B220 and CD11b (Mac-1) (Pharmingen, San

Diego, CA, USA). Analysis was performed using a fluorescein

isothiocyanate-labelled Mac-1, as well as biotin-conjugated

B220 mAbs (Pharmingen, San Diego, CA, USA). Cells were

analysed in a FACS ConvertTM.Software, Facscalibur (Becton

Dickinson, Franklin Lakes, NJ, USA).

Selective body irradiation

The thoracic and abdominal regions of mice were irradiated

with 900 rad using the technique proposed by Novaes and

Brito et al. (unpublished data). Briefly, the animals were held

in a lead device that protects their fore and hind limbs from

irradiation

Cell transfer experiments

Adherent peritoneal cells collected from the peritoneal cavity

of BALB/c mice were cultured for 7 days at 37 �C and pulsed,

for 16 h, with [3H]-tymidine (1 mCi/ml). Free-floating cells

were harvested, washed three times in cold PBS, and aliquots

were adjusted to have 5 · 106 cells in 0.5 ml of PBS. These

aliquots were injected into the peritoneal cavity of naive

BALB/c mice. One hour after cell inoculation, two pockets

were opened in the subcutaneous tissue of the dorsal region of

the animals into which a round glass cover slip was implanted.

Cover slips were removed after 4 days, fixed in Bouin’s fixa-

tive for 2 h, mounted in a glass slide and prepared for his-

toautoradiographic analysis. The slides were coated with K5

autoradiographical emulsion (Ilford Ltd, Ilford, UK) by the

dipping method (Kopriwa & Leblond 1962). After exposure

at 4 �C for 30 days, the radio autographs were developed with

D19b developer (Ilford) fixed and stained with haematoxylin

and eosin.

Reconstitution of CBA/N (Xid) mice with B-1 cells

CBA/N mice were reconstituted with 2.5 · 106 B-1 cells

obtained from cultures of adherent peritoneal cells from

CBA/J mice. Cells were injected by intraperitoneal route,

and 1 week later, groups of CBA/J, CBA/N and CBA/N recon-

stituted animals were implanted with glass cover slips. Cover

slips were removed after 4 days, and the cells on their surface

stained with haematoxylin-eosin and mounted for micro-

scopic examination. The absence of B-1 cells in Xid mice

and irradiated mice and their presence in the peritoneal cavity

of reconstituted animals was monitored by flow cytometry.

B-1 cells and inflammatory giant cell formation 259

� 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 257–265

Statistical analysis

Comparisons among groups were made using ordinary ANOVA.

Statistical significance was assumed at P < 0.05.

Results

B-1 cells are almost absent in the peritoneal cavity of

irradiated mice but present in reconstituted animals

About 21% of the peritoneal cells of BALB/c mice double

stain for IgM and Mac-1 thus characterizing B-1 cells

(Figure 1a). When peritoneal and pleural cavities were irradi-

ated, the percentage of B-1 cells in the peritoneal cavity

dropped to 2% (Figure 1b), and about 12% of B-1 cells

were detected in the peritoneal cavity of mice 7 days after

they were irradiated and reconstituted with B-1 cells

(Figure 1c).

Xid mice have impairment in GC formation

Based on previous observations that B-1 cells migrate to a

non-specific inflammatory lesion (Almeida et al. 2001), Xid

mice, which lack B-1 cells, were used to investigate whether

these cells participate in the kinetics of GC formation. For this

purpose, glass cover slips were implanted into the subcuta-

neous tissue of BALB/c, BALB/c Xid and CBA/J, and CBA/N

mice for 4 days and the mean number of GCs per microscopic

field (10 fields counted in each preparation) evaluated. As

shown in Table 1, BALB/c mice had 6.7 GCs per microscopic

field while 2.6 GCs per microscopic field were detected on

cover slips obtained from BALB/c Xid mice. Similar results

were obtained when CBA/J and CBA/N mice were used.

Approximately, 7.5 GCs per microscopic field were detected

on the surface of cover slips implanted in CBA/J mice whereas

1.5 GCs per microscopic field were observed on the surface of

cover slips implanted into the subcutaneous tissue of CBA/N

mice. Figure 2a and b show histological differences obtained

after 4 days of cover slip implantation in BALB/c and BALB/c

Xid mice.

The kinetics of GC formation in CBA/N and CBA/J mice was

evaluated. As shown in Figure 3, the number ofGCs on the glass

surface increases up to 21 days after cover slip implantation in

mice from the CBA/J strain. Conversely, the maximum number

of GCs per microscopic field was observed after 8 days of cover

slip implantation in CBA/N mice, and was almost absent on

cover slips implanted for 16 and 21 days.

Not only the number of GCs but also the number of nuclei

in these cells were evaluated in Xid and wild-type mice. As

shown in Figure 4a and b, the number of nuclei per GC

varied according to the strain of mice used. GCs formed on

the surface of glass cover slips implanted in mice from the

CBA/J strain had 10 or more nuclei up to 21 days after cover

slip implantation whereas cells with a mean number of five

nuclei were detected up to 8 days of cover slip implantation in

CBA/N mice.

Selective irradiation of BALB/c mice impairs GC

formation

Mice were selectively irradiated with 900 rads and cover slips

were implanted into the subcutaneous tissue of the animals 8

days later. Control mice were not irradiated. The cover slips

were removed 4 days later, and the cells on their surface

analysed. As shown in Figure 5, irradiation of the pleural

and abdominal cavities resulted in less GC formation in the

first 8 days after cover slip implantation. However, irradiated

mice recover their ability to form GCs after 15 and 30 days of

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

21% 1.98% 12%

a b c

Mac-1Mac-1Mac-1

IgM

IgM

IgM

Figure 1 (a),(b),(c) FACS profile of B-1 cells obtained from the peritoneal cavity of normal BALB/c mice (a) from irradiated mice (b) and

irradiated and B-1 cell reconstituted animals (c). Cells were stained with anti-CD11b (Mac-1) and anti-IgM antibodies.

260 C. S. B. Bogsan et al

� 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 257–265

cover slip implantation. Importantly, the migration and

adherence of mononuclear cells on the glass surface of irradi-

ated mice was not impaired (data not shown).

GCs are formed in CBA/N mice reconstituted with B-1

cells

Based on the results described above which suggest the con-

tribution of B-1 cells on GC formation, we decided to recon-

stitute CBA/N mice with cultured B-1 cells and test whether

GC formation could be restored in these animals. In order to

exclude the possible non-specific influence of cell transference

on the phenomenon, normal CBA/J mice were also injected i.p.

with the same number of cells as were injected into CBA/N

mice. Normal CBA/N and CBA/J mice were also used as con-

trols. A week after the cell transference, glass cover slips were

implanted into the subcutaneous tissue of mice from all the

experimental groups, removed after 4 days and the cells on the

glass surface stained with haematoxylin-eosin. As expected, 1.5

cells per microscopic field on cover slips removed from CBA/N

and 7.5 cells per microscopic field on the surface of glass cover

slips removed from CBA/J mice were multinucleated. As shown

in Figure 6, a significant increase in GC formation (34.3 GCs

per microscopic field) was observed on the surface of glass

cover slips implanted in CBA/N reconstituted mice, surpassing

the number of GCs formed on implants obtained from CBA/J

and CBA/N mice. The inoculation of B-1-enriched cell suspen-

sion in CBA/J mice did not influence GC formation, thus

excluding a non-specific effect of these cells in the

phenomenon.

Direct evidence that B-1 cells participate in GC formation

These results provide strong but indirect evidences that B-1

cells participate in the mechanisms of GC formation in a

a

b

Figure 2 (a, b) Giant cells are formed on the surface of glass cover

slips implanted for 4 days into the subcutaneous tissue of CBA/J

mice (a) and are almost absent on glass cover slips implanted, for

the same time, in CBA/N (Xid) mice (b). Mononuclear cell

migration and adherence to the glass surface in CBA/N mice is not

impaired. Haematoxilin-eosin ·400

Num

ber

of G

Cs

per

mic

rosc

opic

fiel

d

Control 8 days 15 days 30 days0

10

20

30

Figure 3 The number of giant cells (GCs) per microscopic field on

cover slips implanted in CBA/J mice increases up to 21 days after

implantation. Conversely, GCs reaches its maximum numbers –

lower that that observed on implants made in CBA/J mice –

8 days after cover slip implantation in CBA/N mice.

Table 1 Mean of the number of giant cells (GCs) per microscopic

field on the surface of glass cover slips implanted for 4 days into

the subcutaneous tissue of different strains of mice

Strain GCs per microscopic field

BALB/c 6.7

BALB/c Xid 2.6*

CBA/J 7.5

CBA/N 1.5*

*P < 0.001

B-1 cells and inflammatory giant cell formation 261

� 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 257–265

foreign-body-induced lesion. Based on previous observations

that B-1b cells migrate from the peritoneal cavity to a non-

specific inflammatory lesion (Almeida et al. 2001), we inves-

tigated whether B-1 cells could fuse to form GCs. For this

purpose, B-1 cells labelled with [3H]-thymidine in vitro were

injected intraperitoneally into BALB/c mice followed by the

implantation of glass cover slips into the subcutaneous tissue

of the animals. The cover slips were removed after 4 days and

prepared for autoradiographic analysis. Results showed that

about 70% of multinuclear GCs had at least one labelled

nucleus, and 17% of the mononuclear adherent cells were

labelled (Figure 7).

IgM is expressed by GCs

As shown in Figure 8, the cytoplasm of a multinucleated cell

on the surface of glass cover slip implanted for 4 days in BALB/

c mice immunostained for both IgM in the cytoplasm and F4/

80 in the outer cell membrane. This pattern of reaction was not

constantly observed. The positive reaction was only obtained

when cells were treated with detergent to permeabilize the cell

membrane, indicating an intracellular location of IgM.

Num

ber

of G

Cs

per

mic

rosc

opic

fiel

d

2–5 6–10 n > 100

10

20

a

b

4 days

8 days

16 days

21 days

Number of nuclei per GC

Num

ber

of G

Cs

per

mic

rosc

opic

fiel

d

2–5 6–10 n > 100.0

2.5

5.0

7.5

10.0

4 days

8 days

16 days

21 days

Figure 4 (a,b) The number of nuclei per giant cell was

evaluated. Figure 4a shows that cells with 10 or more nuclei

were observed on the surface of glass cover slips implanted for up

to 21 days into the subcutaneous tissue of mice from the CBA/J

strain. However, cells with no more then five nuclei were

observed on the surface of cover slips implanted for 8 days into

the subcutaneous tissue of CBA/N mice.

Num

ber

of G

Cs

per

mic

rosc

opic

fiel

d

Control 8 days 15 days 30 days0

10

20

30

Figure 5 The selective irradiation of CBA/J mice induces

impairment in giant cell formation up to 8 days of cover slip

implantation. This effect is progressively reversed after 15 and 30

days of irradiation.

Num

ber

of G

Cs

per

mic

rosc

opic

fiel

d

Control B-1 transferred0

10

20

30

40 CBA/J

CBA/N*

Figure 6 CBA/N mice were reconstituted with B-1 cells obtained

from cultures of adherent CBA/J peritoneal cells. CBA/J mice

were also injected with the same cell suspension. Results show

that transference of B-1 cells to CBA/J mice does not influence the

number of giant cells (GCs) formed on the surface of implanted

cover slips. Conversely, the cell transference to CBA/N mice

restored their ability to form GCs.

262 C. S. B. Bogsan et al

� 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 257–265

Discussion

The origin, properties and fate of foreign body and other types

of GCs are fragmentary. Here, we bring direct evidences that

GCs induced by the implantation of glass cover slips into the

subcutaneous tissue of mice is dependent on the participation

of B-1 cells.

The Xid (Amsbaugh et al. 1972; Scher 1982) share features

with the severe immunodeficiency linked with the X chromo-

some that occurs in man. In Xid mice, the number of B-2 cells

(conventional B cells) is only slightly reduced. Here, we con-

firm that these mice lack or have only small numbers of cells

(Riggs et al. 2003) with the characteristics of B-1b cells,

expressing IgMlo, IgDhi, MAC-1 and B-220 antigens (Scher

1982; Hardy et al. 1983). Another subset of B-1 cells, the B-1a

cells have all the characteristics of B-1b cells but are also

CD5+. B-1a cells are also absent in Xid mice (Iacomini &

Imanishi-Kari 1992).

The hypothesis that GCs are formed by the concurrence of

two populations of macrophages one ‘old’ and another newly

arrived from circulation, as proposed by Mariano & Spector

(1974), and the recent demonstration by our group that B-1

cells exit coelomatic cavities and migrate to an inflammatory

site where they can transform into a novel type of mono-

nuclear phagocyte not related to the monocyte-derived macro-

phage (Almeida et al. 2001), led us to investigate the possible

role that B-1 cells might play in GC formation.

In a first set of experiments, we analysed the kinetic of GC

formation in wild and Xid mice. Results clearly show that Xid

mice have impairment in GC formation. In addition, there are

also fewer nuclei in GCs formed in Xid mice when compared

with preparations obtained from wild mice. Further indirect

evidence of the participation of B-1 cells in the phenomenon

was obtained with the irradiation and cell transfer experiments.

B1 cells are radiosensitive (Almeida et al. 2001), and when the

peritoneal and pleural cavities of the animals were irradiated,

GC formation was drastically reduced. Further, the reconstitu-

tion of Xid mice with B-1 cells from adherent peritoneal cells

fromwildmice resulted in an increase in the number of GCs that

surpassed that observed in control groups.

The transfer experiments with [3H]-thymidine-labelled B-1

cells and the immunostaining for IgM expression analysed by

confocal microscopy brought further evidence that B-1 cells

participate in the mechanisms of GC formation in this model.

The transfer experiments with [3H]-thymidine-labelled B-1

cells showed clearly that not only mononuclear cells with a

macrophage-like morphology but also a large proportion of

GCs had labelled nuclei thus indicating that B-1 cells migrate

from the peritoneal cavity to the inflammatory milieu. Almost

all the GCs in these preparations had from one to five labelled

Figure 8 Cells on the surface of glass cover slips implanted for

4 days into the subcutaneous tissue of BALB/c mice were

labelled with monoclonal antibodies anti-F4/80 (red), and

immunoglobulin (IgM) (green) and DAPI (blue) and analysed

by confocal microscopy. A typical foreign-body giant cell (GC)

expressing IgM in the cytoplasm and F4/80 in the outer

membrane is shown. F4/80+ mononuclear cells are in intimate

contact with the GC. Arrows indicate mononuclear cells

expressing IgM.

Figure 7 [3H]-thymidine was added to 7-day cultures of

adherent peritoneal cells from BALB/c mice. Labelled cells

were collected after 16 h and transferred to the peritoneal

cavity of naive BALB/c mice. One hour after cell transfer-

ence, glass cover slips were implanted into the subcutaneous

tissue of the animals and removed after 4 days for auto-

radiographic analysis. Silver grains are observed on the

nuclei of a typical Langerhans giant cell (GC). Haematoxilin-

eosin ·1000

B-1 cells and inflammatory giant cell formation 263

� 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 257–265

nuclei. The non-specific labelling of these cells is difficult to be

admitted, because the B-1 cells were labelled in vitro, thor-

oughly washed before transference and analysed after 4 days.

The demonstration that GCs have IgM in their cytoplasm

also supports the view that B-1 cells participate in the genesis

of these cells although endocytosis of IgM should be excluded.

This hypothesis can be ruled out on the basis that B-1 cells

secret large amounts of IgM in vivo (Stall et al. 1992) and in

vitro (Almeida et al. 2001). Second, it has been demonstrated

in our laboratory that these cells have a promiscuous expres-

sion of myeloid and lymphoid transcription factors, loosing

the expression of lymphoid factors as they differentiate into a

mononuclear phagocyte. As analysed by immunostaining and

confocal microscopy, B-1 cells loose IgM expression in the

course of differentiation keeping the expression of myeloid

markers (Popi et al. 2004). The same pattern of immunostain-

ing for IgM was observed in cells on the cover slip surface.

Although most of the mono and multinucleated cells stained

for myeloid markers (Mac-1, F4/80) the expression of both

IgM/F4/80 was detected in a fewer number of cell. If endo-

cytosed, it should be expected IgM to be present in the cyto-

plasm of the majority of cells on the glass surface.

Although these data demonstrate the participation of B-1

cells in GC formation, it remains to be seen whether GCs

are solely B-1 derived or whether they result from fusion of

B-1 derived mononuclear phagocytes and monocyte-derived

macrophages. This question is an issue under investigation

in our laboratory. It also remains to be demonstrated

whether GC formation in different pathologies are depend-

ent on B-1 cell participation. If so, it opens the possibility

not only to better understand the biologic significance of

the intriguing phenomenon of phagocyte multinucleation in

vivo but also to investigate the participation of adaptive

immunocompetent cells in determining the course of non-

immune inflammation.

Acknowledgements

This paper is dedicated to the memory of W.G. Spector. We

are indebted to Prof Telma Zorn for the preparation of the

historadiograms. We are supported by grant from Fundacao

de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), no

99/11144–4

References

Abrahao T.B., Freymuller E., Mortara R.A., Lopes J.D.,MarianoM.

(2003) Morphological characterization of mouse B-1 cells.

Immunobiology. 208, 401–411.

Almeida S.R., Aroeira L.S., Frymuller E. et al. (2001) Mouse B-1

cell-derived mononuclear phagocyte, a novel cellular compo-

nent of acute non-specific inflammatory exudate. Int.

Immunol. 13, 1193–1201.

Amsbaugh D.F., Hansen C.T., Prescott B., Stashak P.W.,

Barthold D.R., Baker P.J. (1972) Genetic control of the anti-

body response to type 3 pneumococcal polysaccharide in mice.

I. Evidence that an X-linked gene plays a decisive role in

determining responsiveness. J. Exp. Med. 136, 931–949.

Anderson J.M. (2000) Multinucleated giant cells. Curr. Opin.

Hematol. 7, 40–47.

BorrelloM.A., Palis J., Phipps R.P. (2001) The relationship of CD5+

B lymphocytes to macrophages: insights from normal bipheno-

typic b/macrophage cells. Int. Rev. Immunol. 20, 137–155.

Brand I., Buoen L.C., Brand K.G. (1977) Foreign-body tumors of

mice: strain and sex differences in latency and incidence.

J. Natl. Cancer. Inst. 58, 1443–1447.

Chambers T.J. (1977) Failure of altered macrophage surface to

lead to the formation of polykaryons. J. Pathol. 122, 185–189.

Chambers T.J. (1978) Multinucleate giant cells. J. Pathol 126,

125–148.

Chambers T.J. & Spector W.G. (1982) Inflammatory giant cells.

Immunobiology 161, 283–289.

Enelow R.I., Sullivan G.W., Carper H.T., Mandell G.L. (1992a)

Cytokine-induced human multinucleated giant cells have

enhanced candidacidal activity and oxidative capacity com-

pared with macrophages. J. Infect. Dis. 166, 664–668.

Enelow R.I., Sullivan G.W., Carper H.T., Mandell G.L. (1992b)

Induction of multinucleated giant cell formation from in vitro

culture of human monocytes with interleukin-3 and interferon-

gamma: comparison with other stimulating factors. Am. J.

Respir. Cell. Mol. Biol. 6, 57–62.

Fais S., Burgio V.L., Capobianchi M.R., Gessani S., Pallone F.,

Belardelli F. (1997) The biological relevance of polykaryons in

the immune response. Immunol. Today. 18, 522–527.

Fais S., Burgio V.L., Silvestri M., CapobianchiM.R., Pacchiarotti A.,

Pallone F. (1994) Multinucleated giant cells generation

induced by interferon-gamma. Changes in the expression and

distribution of the intercellular adhesion molecule-1 during

macrophages fusion and multinucleated giant cell formation.

Lab. Invest. 71, 737–744.

Gillman T. & Wright L.J. (1966) Probable in vivo origin of multi-

nucleated giant cells from circulating mononuclears. Nature.

209, 263–265.

Hardy R.R., Hayakawa K., Parks D.R., Herzenberg L.A. (1983)

Demonstration of B-cell maturation in X-linked immunodefi-

cient mice by simultaneous three-colour immunofluorescence.

Nature. 306, 270–272.

Harris H. (1970) Cell Fusion. pp. 108. Ipswich: Oxford

University Press.

Hassan N.F., Kamani N., Meszaros M.M., Douglas S.D. (1989)

Induction of multinucleated giant cell formation from human

264 C. S. B. Bogsan et al

� 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 257–265

blood-derived monocytes by phorbol myristate acetate in in

vitro culture. J. Immunol. 143, 2179–2184.

Iacomini J. & Imanishi-Kari T. (1992) The effect of an immuno-

globulin mu transgene on B cell maturation. Eur. J. Immunol.

22, 745–751.

Kazazi F., Chang J., LopeZ. A., Vadas M., Cunningham A.L.

(1994) Interleukin 4 and human immunodeficiency virus sti-

mulate LFA-1-ICAM-1-mediated aggregation of monocytes

and subsequent giant cell formation. J. Gen. Virol. 75 (10),

2795–2802.

Keymeulen K. & Dillemans B. (2004) Epitheloid angiosarcoma of

the splenic capsula as a result of foreign body tumorigenesis. A

case report. Acta. Chir. Belg. 104, 217–220.

Kopriwa B. & Leblond C. (1962) Improvements in the coating

technique of radioautography. J. Histochem. Cytochem. 10,

269–284.

Kreipe H., Radzun H.J., Rudolph P. et al (1988) Multinucleated

giant cells generated in vitro. Terminally differentiated macro-

phages with down-regulated c-fms expression. Am. J. Pathol.

130, 232–243.

Mariano M. & Spector W.G. (1974) The formation and proper-

ties of macrophage polykaryons (inflammatory giant cells).

J. Pathol. 113, 1–19.

McInnes A. & Rennick D.M. (1988) Interleukin 4 induces cul-

tured monocytes/macrophages to form giant multinucleated

cells. J. Exp. Med. 167, 598–611.

McNally A.K. & Anderson J.M. (1995) Interleukin-4 induces

foreign body giant cells from human monocytes/macrophages.

Differential lymphokine regulation of macrophage fusion leads

to morphological variants of multinucleated giant cells. Am. J.

Pathol. 147, 1487–1499.

Metchnikoff E. (1968) Lectures on the Comparative Pathology of

Inflammation. New York: Dover Publications, Inc.

Most J., Neumayer H.P., Dierich M.P. (1990) Cytokine-induced gen-

eration of multinucleated giant cells in vitro requires interferon-

gamma and expression of LFA-1. Eur. J. Immunol. 20, 1661–1667.

Most J., Spotl L., Mayr G., Gasser A., Sarti A., Dierich M.P.

(1997) Formation of multinucleated giant cells in vitro is

dependent on the stage of monocyte to macrophage matur-

ation. Blood. 89, 662–671.

Nagasawa H., Miyaura C., Abe E., Suda T., Horiguchi M. (1987)

Fusion and activation of human alveolar macrophages induced

by recombinant interferon-gamma and their suppression by

dexamethasone. Am. Rev. Respir. Dis. 136, 916–921.

Papadimitriou J.M. & Robertson T.A., Walters M.N. (1975) An

analysis of the phagocytic potential of multinucleate foreign

body giant cells. Am. J. Pathol. 78, 343–358.

Papadimitriou J.M. & Walters M.N. (1979) Macrophage poly-

karya. CRC. Crit. Rev. Toxicol. 6, 211–255.

Popi A.F., Lopes J.D., Mariano M. (2004) Interleukin-10 secreted

by B-1 cells modulates the phagocytic activity of murine macro-

phages in vitro. Immunology. 113, 348–354.

Postlethwaite A.E., Jackson B.K., Beachey E.H., Kang A.H.

(1982) Formation of multinucleated giant cells from human

monocyte precursors. Mediation by a soluble protein from

antigen-and mitogen-stimulated lymphocytes. J. Exp. Med.

155, 168–178.

Riggs J., Howell K., Matechin B., Matlack R., Pennello A.,

Chiasson R. (2003) X-chromosome-linked immune-deficient

mice have B-1b cells. Immunology. 108, 440–451.

Ryan G.B. & Spector W.G. (1970) Macrophage turnover in

inflamed connective tissue. Proc. R. Soc. Lond. B Biol. Sci.

174, 269–292.

Scher I. (1982) The CBA/N mouse strain: an experimental model

illustrating the influence of the X-chromosome on immunity.

Adv. Immunol. 33, 1–71.

Spector W.G. & Lykke A.W. (1966) The cellular evolution of

inflammatory granulomata. J. Pathol. Bacteriol. 92, 163–167.

Stall A.M., Adams S., Herzenberg L.A., Kantor A.B. (1992)

Characteristics and development of the murine B-1b (Ly-1 B

sister) cell population. Ann. N Y Acad. Sci. 651, 33–43.

Takashima T., Ohnishi K., Tsuyuguchi I., Kishimoto S.

(1993) Differential regulation of formation of multi-

nucleated giant cells from concanavalin A-stimulated human

blood monocytes by IFN-gamma and IL-4. J. Immunol. 150,

3002–3010.

Weinberg J.B., Hobbs M.M., Misukonis M.A. (1987)

Recombinant human gamma-interferon induces human mono-

cyte polykaryon formation. Proc. Natl. Acad. Sci. USA. 81,

4554–4557.

B-1 cells and inflammatory giant cell formation 265

� 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 257–265