Mouse B-1 cell-derived mononuclear phagocyte, a novel cellular component of acute non-specific inflammatory exudate

Sandro Rogério Almeida, Luiz Stark Aroeira2, Edna Frymuller1, Maria Ângela Amorim Dias, Cristina Stewart Bittencourt Bogsan, José Daniel Lopes and Mario Mariano

Discipline of Immunology, Department of Microbiology, Immunology and Parasitology, and
1 Center of Electron Microscopy, Federal University of São Paulo, Rua Botucatu 862, 04023-9000, São Paulo, Brazil
2 Institute of Nuclear and Energetic Research, PBR–Travessa R, no. 400, 05508-9000 São Paulo, Brazil

Correspondence to: M. Mariano, E-mail: mariomu{at}aol.com.br


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
At least three B cell subsets, B-1a, B-1b and B-2, or conventional B cells are present in the mouse periphery. Here we demonstrate that B-1 cells spontaneously proliferate in stationary cultures of normal adherent mouse peritoneal cells. B-1 cells were characterized by morphology, immunohistochemistry and flow cytometry. IgM was detected in the supernatants of these cultures. We demonstrated that the major cell population analyzed expresses the B-1b phenotype. When these cells were transferred to a new culture, a large proportion of them adhere to the plastic surface, and spread as bipolar cells endowed with the capacity to phagocytose via Fc and mannose receptors. Flow cytometry analysis of these adherent cells demonstrated that the great majority of them share both B-220 and Mac-1 antigens. Nevertheless, 45% of them were exclusively Mac-1+. Finally, when they were labeled in vitro with [3H]thymidine and transferred to the peritoneal cavity of naive mice, they migrate to a non-specific inflammatory focus induced by a foreign-body implant. These data demonstrate that B-1 cells, mainly B-1b cells, not only proliferate and differentiate into a mononuclear phagocyte in vitro, but also that they exit the peritoneal cavity and migrate to a non-specific inflammatory milieu.

Keywords: inflammation, lymphocyte, macrophage, peritoneal cells, phagocyte, phagocytosis


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The demonstration that macrophages derive from circulating blood monocytes (1) and that macrophages in an inflammatory milieu also have a monocytic origin (2,3) resulted in the Mononuclear Phagocytic System concept (4). So far, it has not been demonstrated that macrophages can originate from other cell lines or differentiate into subsets with distinct function (5), as has been demonstrated to occur with lymphocytes. However, well-documented reports over recent years showed evidence that both the B lymphoid and myeloid lineages share an unexpectedly close relationship. This is based on studies demonstrating that a number of mice (6,7) and human (8,9) B-1 malignancies can generate phagocytic descendants exhibiting macrophage-like characteristics. The same phenomenon has been described for normal mouse B-1a cells (CD5+) when they were co-cultured with fibroblasts. The authors called these cells bi/phenotypic macrophages (10). Recently, the same group demonstrated that B/macrophage cells express COX-1, and up-regulate COX-2 expression and prostaglandin E2 production in response to pro-inflammatory signals. They have also confirmed the existence of B-1 cells in the normal peritoneal cavity of mice (11). However, despite the large volume of information concerning the origin (1215), properties (6,13,16,17) and participation of these cells in normal (12,18,19) and pathologic conditions (9,2026), the demonstration of whether the generation of mononuclear phagocytes from B-1 cells is one of the fates of these cells in vivo remains to be established.

Herein we demonstrate that B-1 cells, mainly B-1b cells, proliferate in primary cultures of normal adherent mouse peritoneal cells and transform into a novel type of mononuclear phagocyte, not related with the monocyte-derived macrophage. Further, that these cells are endowed with the ability to exit the peritoneal cavity and migrate to a site of a foreign body-induced acute inflammation where they differentiate into macrophage-like cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
A/Sn, BALB/c and BALB/c Xid female mice, 8–12 weeks old, were used. Mice were obtained from the animal facilities of the Department of Immunology, University of São Paulo, Brazil.

Adherent peritoneal cells
Peritoneal cells from normal mice were collected after peritoneal washing with PBS and isolated by selective adherence to glass Petri dishes for 2 h at 37°C. Adherent cells were detached by scraping with a silicone rubber, washed and suspended in RPMI 1640 (Sigma, St Louis, MO) complete medium containing 10% heat-inactivated FCS (Gibco/BRL, Gaithersburg, MD). In order to rule out the influence of FCS as the source of the observed results, FCS from three different brands were tested with similar results. The cells were plated in 24-well plates at a concentration of 2x106 cells/well and incubated at 37°C in an atmosphere of 5% CO2 for 7 days. During this period, the culture medium was not changed.

Proliferative response assays
Adherent peritoneal cells were isolated as described above. Adherent cells in complete medium (RPMI 1640 plus 10% FCS) were plated at a concentration of 2x106 cells/well in 96-well plates. Cells were cultured for 2, 4 and 7 days without changing the culture medium. Sixteen hours before harvesting the free-floating cells in each experiment, the cultures were pulsed with [3H]thymidine (1 µCi/ml). The cells were collected with an automated cell harvester and incorporated radioactivity measured in a Beckman liquid scintillation counter. Contamination of the cultures with lipopolysaccharide (LPS) was monitored using the Limulus amebocyte lysate test (Endosafe LAL, Charles River, Charleston, SC). Maximal concentration of LPS detected in the cultures was always <0.1 ng LPS/ml.

Mice and cell irradiation
Mice from the A/Sn strain were lethally irradiated (700 rad) in a Nordion Gammacell, and, 2 days later, cells from their peritoneal cavities were collected and cultured as described above. In some experiments, adherent peritoneal cells from normal mice were irradiated as above and cultured for 7 days.

Scanning electron microscopy
Cells cultured on the surface of glass cover slips were fixed for 1 h in 2.5% glutaraldehyde plus 2% p-formaldehyde in sodium cacodylate buffer, washed in cacodylate buffer and fixed in 2% buffered osmium tetroxide for 1 h. The specimens were dehydrated in graded ethanol solutions and critical-point dried. After coating with gold the samples were examined in a JEOL JSM 5.300 scanning electron microscope.

Immunohistochemistry for MRP-14 detection
Monospecific rabbit polyclonal antibodies anti-human MRP-14, that cross-react with murine MRP-14, or rat mAb anti-mouse MRP-14 were used to detect the expression of the protein in adherent and non-adherent cells. These preparations were fixed in cold methanol and immunostained using peroxidase-conjugated goat anti-rat or anti-rabbit IgG as described (27). Professor Clemens Sorg (Institute of Dermatology, University of Münster, Germany) kindly provided the antibodies.

Analysis of non-adherent cell phenotypes
Two- or three-color flow cytometry was used to determine the phenotype of non-adherent cells in stationary 7-day cultures of adherent normal peritoneal cells. The cultures were washed with PBS plus 10% FCS, and 20,000 events were stained with mAb directed to mouse CD45/B-220 (RA3-6B2), CD5 (53-7.3), Mac-1 (MA/70) and IgM. Staining for flow cytometry analysis was performed using an phycoerythrin-labeled anti-IgM, FITC-labeled anti-CD-5 and Mac-1, as well as biotin-conjugated anti-B-220 mAb (PharMingen, San Diego, CA). Two- or three-color staining was performed using the antibodies described above and analyzed in a FACSCalibur (Becton Dickinson, Mountain View, CA).

IgM secretion in vitro
The presence of IgM in the supernatant of cultures of normal peritoneal cells was analyzed after 2, 4 and 7 days of culture. IgM concentration in these samples was performed using the mouse isotyping panel from BioRad (Hercules, CA). Immunoplates (96-well; Costar, Corning Costar Japan, Tokyo, Japan) were coated for 1 h with the supernatants and washed in PBS with 0.05% Tween for 3 times. Remaining binding sites were blocked with 1% BSA in PBS for 1 h at room temperature. Rabbit anti-mouse IgM was added and after 1 h at room temperature the plates were washed with PBS with 0.05% Tween. The presence of IgM was detected with anti-rabbit horseradish peroxidase at 1:3000 dilution. Absorbance at 405 nm was determined using a Multiskan (Labsystem Multiskan MR4000 Dynatech, Chantilly, VA) MCC/340 II reader. The amount of IgM in the supernatants was evaluated using a standard curve produced with purified mouse IgM.

Re-culture of non-adherent cells
After 7 days of culture, non-adherent cells were harvested, washed and re-cultured in fresh medium (RPMI 1640 plus 10% FCS) in 24-well tissue culture plates containing round glass coverslips in the bottom. Part of these cells, before addition to the plates, was treated with a goat polyclonal antibody anti-mouse IgM (10 µg/ml) (Sigma) for 40 min at 4°C followed by addition of rabbit complement and incubation for 30 min at 37°C. Following this treatment, cells were analyzed by double-color flow cytometry using anti-B-220–phyceorythrin and anti-Mac-1–FITC mAb.

Phagocytic assays
Sheep red blood cells (SRBC) were opsonized by a 1 h incubation at 37°C in 2 ml of PBS containing a 1/100 dilution of rabbit anti-SRBC IgG followed by 1 h incubation at 37°C. Aggregates were allowed to settle and opsonized SRBC were incubated with 2x104 re-cultured adherent cells in 24-well tissue culture plates containing round glass coverslips in the bottom, for 2 h at 37°C. Cultures were washed twice with PBS and non-internalized SRBC were lysed by a brief hypotonic shock with hemolytic buffer. Subsequently, coverslips were vigorously washed in PBS, the cells fixed in 2% glutaraldehyde, and phagocytosis was analyzed by phase-contrast and scanning electron microscopy. The adherent cells were cultured as described above, together with 20 µl of a 2% suspension of Saccharomyces cerevisae zymozan (Sigma) in PBS and incubated for 1 h at 37°C. The cultures were then washed, fixed and phagocytosis analyzed by phase-contrast microscopy.

Cell-transfer experiments
Adherent peritoneal cells were cultured for 7 days at 37°C and pulsed, for 16 h, with [3H]thymidine (1 µCi/ml). Free-floating cells were harvested, washed for 3 times in cold PBS and aliquots were adjusted to have 5x106 cells in 0.5 ml of PBS. These aliquots were injected into the peritoneal cavity of naive mice. One hour after cell inoculation, two pockets were opened in the s.c. tissue of the dorsal region of the animals into which a round glass coverslip was implanted (2,3). Coverslips were removed after 24 and 48 h, fixed in Bouin's fixative for 6 h, mounted in a glass slide, and prepared for histoautoradiographic analyses. The slides were coated with K5 radioautographical emulsion (Ilford, Essex, UK) by the dipping method (28). After exposure at 4°C for 30 days, the radioautographs were developed with D19b developer (Ilford), fixed and stained with hematoxylin & eosin.

IgM expression by inflammatory cells
Glass cover slips were implanted into s.c. tissue of BALB/c mice and removed after 4 days. The glass cover slips were washed in PBS and fixed in 3.5% formalin in PBS for 30 min. The coverslips were washed in PBS and free radicals blocked with PBS containing 0.1% BSA (Sigma). Cell membranes were permeabilized by treatment with 0.01% Triton X-100 for 30 min. Polyclonal goat antibody anti-mouse IgM (µ chain specific) (Sigma) was diluted 1:50 in PBS/BSA/Triton and incubated with the glass coverslips for 1 h at room temperature. After three washes in PBS, glass cover slips were incubated with FITC-conjugated rabbit F(ab')2 anti-goat IgG (H + L) (Southern Biotechnology Associates, Birmingham, AL) also diluted in PBS/BSA/Triton in the presence of 1 µM DAPI to label DNA-rich structures. After a further 1 h incubation, glass cover slips were washed 3 times in PBS, mounted in 0.1 M glycerol (Quimibrás Indústria Química, Sao Paulo, Brazil) and sealed with nail varnish. Samples were imaged on BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. A x40 NA 1.2 Plan-Apochromatic water immersion objective with Nomarski differential interference contrast optics was used. Kalman averaging of at least 15 frames (512x512 pixels) using a maximum aperture (pinhole) of 2.0 mm collected all images. The collected DIC images were sharpened with a minimum setting using BioRad Lasersharp 1024 software version 2.1a. Prints were generated by dye-sublimation on a Codonics NP1600 printer.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
`Small round cells' that proliferate in stationary cultures of adherent peritoneal cells, are radiosensitive and secrete IgM
Adherent peritoneal cells were cultured for ~7 days without changing the culture medium. Under these experimental conditions a large number of free-floating small round cells with lymphoid characteristics were observed in the culture medium. They were highly refringent when observed by phase-contrast microscopy (Fig. 1AGo). As shown in (Fig. 1BGo), these cells present minute membrane projections and can adhere to the outer cell membrane of typical adherent macrophages.



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Fig. 1. Peritoneal cells from normal mice were allowed to adhere to glass coverslips in 24 plastic wells for 2 h at 37°C. The cultures were than vigorously washed with PBS, supplemented with RPMI 1640 plus 10% FCS and maintained for 7 days without changing the culture medium. In addition to typical adherent macrophages, small, highly refringent and free-floating cells (A) started to appear in the culture medium after 3–4 days of culture (phase contrast microscopy, x200). Observed under scanning electron microscopy (B), the small round cells that varied in size were seen as adherents to the cell membrane of typical adherent macrophages (x3500).

 
They multiply in vitro as demonstrated by [3H]thymidine incorporation (Fig. 2Go). Ig of the IgM isotype was detected in the culture medium with maximum values in 7-day cultures (Fig. 3Go). When either mice or adherent cells were irradiated (700 rad) before the establishment of the cultures, the small and refringent cells no longer appeared in the culture medium.



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Fig. 2. Morphological observation of normal peritoneal cell cultures suggested that the observed small round cells were proliferating in culture. [3H]Thymidine was added after 2, 4 and 7 days of culture at the concentration of 1 µCi/ml 16 h before the cells were harvested. Results show crescent isotope incorporation by the cells, thus demonstrating their proliferative ability in vitro.

 


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Fig. 3. Aliquots of the supernatant of cultures of adherent peritoneal cells were collected, centrifuged and used for the determination of the concentration of IgM, with the aid of a commercial mouse isotyping panel. The maximum concentration of IgM was detected in 7-day cultures.

 
`Small round cells' have a B-1 cell phenotype
When non-adherent cells were analyzed by flow cytometry, three distinct cell populations that differed in their size were observed. These populations corresponded in size to the size of red blood cells, lymphocytes and macrophages respectively. In order to characterize the cells present in these three populations, each of them was electronically selected, and CD5, Mac-1 and IgM expression evaluated by flow cytometry. Results showed that virtually all small cells were IgM+ and Mac-1+, while a small proportion of them stained with anti-IgM and anti-CD5 antibodies. About 34% of the medium-sized cell population were IgM+, and 22% were, concomitantly, Mac-1+ and IgM+. A low proportion of these cells (~3%) also expressed the CD5 antigen (Fig. 4AGo). The medium-sized cell population contained a CD5+ population that did not express IgM. However, this population did express CD3, indicating that they were T cells (data not shown). The majority of the large cells were IgM+ and Mac-1+, with a few of them being concomitantly CD5+. In this population we also found a T cell population that stained only with anti-CD5 antibody (data not shown). Based on results obtained with anti-IgM antibody, the co-expression of B-220, another B cell marker, as well as Mac-1 or CD5, was analyzed. Virtually all the small cells were MAC-1+ and B-220+, and ~20% of B-220+ cells also stained with anti-CD5 antibodies (Fig. 4BGo). T cells (CD5+ and/or CD3+) were not detected in the small cell population (data not shown). The medium-sized cell population had ~40% of B-220+ cells out of which 32% were B-220+ and Mac-1+ double-positive. Approximately 4% of these cells were B-220+ and CD5+. In the large cell population the frequency of B-220+ cells was ~93% of the total, with ~25% being CD5+. Together, these results indicate that B-1 cells (IgM+, B-220+, CD5+ and Mac-1+) proliferate in stationary cultures of normal mouse peritoneal cells. Further, that the great majority of B-1 cells present in the culture medium are B-1 b cells (IgM+, B-220+, Mac-1+ and CD5). Additional evidence for the presence and proliferation of B-1 cells in the cultures was obtained when peritoneal cells from Xid mice, which lack B-1 cell precursors (29), were tested. Under these conditions, the large population of small and refringent cells no longer appeared in the culture medium after 7 days of culture.



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Fig. 4. Free-floating cells were separated from adherent peritoneal cell cultures (7 days) and analyzed by flow cytometry as described in Methods. Cells were double stained for IgM and Mac-1 (A, upper panel), and electronically gated as small, medium and large sizes. A large proportion of these cells (95%) was both IgM+ and Mac-1+. A small proportion of them (<5%) was composed of IgM+ and CD5+ cells (A, lower panel). Cells stained for B-220 and Mac-1 (B, upper panel) were also double-positive, and a small proportion (<5%) was both B-200+ and CD5+ cells (lower panel).

 
Re-culture of B-1 cells generate macrophage-like cells
When non-adherent cells from 7-day cultures were collected, washed and re-cultured in fresh medium, a proportion of the small round cells adhered to the plastic and underwent characteristic transformations. After 24 h of culture, these cells became larger and projected pseudopods from two poles of the cell. These pseudopods became more prominent and typical bipolar cells appeared. Their nucleus, uniquely, remained oval with a condensed chromatin (Fig. 5Go). Evidence that these adherent cells originate from B-1 cells and not from any other cell population was obtained. First, when the non-adherent cell suspension was treated with an anti-IgM antibody plus complement, the bipolar adherent cells no longer appeared in the cultures. Second, the cytophotometric analysis of the adherent cells (Fig. 6Go) showed that they could be separated into three cell populations by their size. A high proportion of these populations was IgM+ and Mac-1+ and B200+ and Mac-1+. Further, ~45% of them no longer expressed B-220 molecules on their surface, becoming single Mac-1+ cells.



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Fig. 5. As shown in Fig. 4(A and BGo), >95% of the small round cells that proliferate in cultures of adherent mouse peritoneal cells could be classified as B-1b cells (IgM+, B-220+, Mac-1+, CD5). When these cells were collected from 7-day cultures, washed for 3 times in PBS and re-cultured in RPMI 1640 plus 10%FCS, they adhered to the plastic surface and spread to become a macrophage-like cell. Phase-contrast microscopy (x400).

 


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Fig. 6. Adherent macrophage-like cells obtained as described in Fig. 5Go were removed from the plastic surface with the aid of a rubber policeman and analyzed by flow cytometry. Cells were double-stained for B-220 and Mac-1 or IgM and Mac-1, and electronically gated as small, medium and large sizes. A high proportion of these cells was B-220+ and Mac-1+ cells. About 45% of them no longer expressed B-220 molecules, becoming single Mac-1+ cells. Similar results were obtained when IgM and Mac-1 were used.

 
Fc and mannose receptors are expressed by B-1-derived macrophage-like cells
The possible expression of both Fc and mannose receptors in these cells was investigated. Results showed that IgG opsonized sheep red blood cells avidly adhere to the cell membrane of B-1-derived mononuclear phagocytes (Fig. 7AGo). When external adherent red blood cells were lysed by a hypotonic solution, the internalization of the particle could be observed (Data not shown). It has been demonstrated that zymozan particles are phagocytosed by macrophages via the mannose receptor (30). B-1-derived mononuclear phagocytes also avidly phagocytose zymozan particles, thus suggesting that they also express the mannose receptor on their surface membrane (Fig. 7BGo).



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Fig. 7. The possible phagocytic activity of the macrophage-like cells was analyzed. Free-floating cells obtained from 7-day cultures of normal mouse peritoneal cells were collected and re-cultured. The phagocytic ability of these cells via Fc receptor was tested. SRBC opsonized with a rabbit polyclonal antibody anti-SRBC were incubated with B-1 cell-derived macrophage-like cells. Results show that these cells avidly phagocytose opsonized red blood cells (A) and zymozan particles (B).

 
B-1 cells exit from the peritoneal cavity and traffic to an inflammatory focus
Based on previous observations that B-1 cells (B-1a) injected into the peritoneal cavity of SCID mice had the ability to migrate to the lamina propria of the intestine of mice and transform into IgA-secreting cells (19), we decided to investigate whether B-1 cells could migrate from the peritoneal cavity to an acute inflammatory focus. For this purpose, B-1 cells were grown in vitro and labeled with [3H]thymidine as described. Free-floating cells were collected from cultures and injected into the peritoneal cavity of normal mice (5x106 cells/animal). One hour after the cells were injected, a round glass coverslip was implanted into the s.c. tissue of the animals, as previously described (2). The coverslips were removed 24 and 48 h after implantation, and prepared for histoautoradiographic analysis. Few cells with labeled nuclei were observed on the surface of coverslips implanted for 24 h. However, the percentage of cells with labeled nuclei on the surface of coverslips implanted for 48 h increased to ~10–14% (Fig. 8Go), indicating that labeled cells injected into the peritoneal cavity migrated to the foreign body-induced acute inflammatory milieu. Labeled cells were adherent to the glass surface presenting an oval or horseshoe-shaped nucleus with a large cytoplasm resembling the typical morphology of monocyte-derived macrophages. Not a single polymorphonuclear cell was labeled, thus excluding the possibility that proliferating bone marrow cells could incorporate [3H]thymidine from cells labeled in cultures.



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Fig. 8. [3H]Thymidine (1 µCi/ml) was added to 7-day cultures of normal peritoneal cells and the free-floating cells were harvested after 17 h. Cells were washed in PBS (3 times) and adjusted for the concentration of 5x106 cells in 0.5 ml. These aliquots were injected into the peritoneal cavity of syngeneic mice. One hour later, the injected animals were implanted with round glass coverslips into the dorsal s.c. tissue. Coverslips were removed after 24 and 48 h after implantation and prepared for histoautoradiographic analysis. As shown, labeled cells (10–14%) with a morphology similar to macrophages were observed on the surface of the implanted glass coverslips. Hematoxylin & eosin (x400).

 
Inflammatory macrophage-like cells express IgM
As shown in Fig. 9Go, it was possible to characterize macrophage-like cells on the surface of coverslips implanted for 4 days into the s.c. tissue of mice expressing IgM in their cytoplasm. Interesting to note that positive reaction was observed only when the cells were treated with Triton. This observation indicates that IgM is not expressed on the outer membrane of these cells.



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Fig. 9. Based on the demonstration that B-1 cells migrate to a non-specific site of inflammation we decided to investigate the existence of cells expressing IgM in the coverslip model. Cells on the surface of coverslips were analyzed by Nomarski differential interference contrast imaging (A), and overlay of nuclei labeling with DAPI (blue) and IgM (green) (B). Using these methodologies it was possible to detect a small number of cells with a macrophage-like morphology expressing this type of Ig in their cytoplasm. Magnification bar in µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Data herein described support the conclusion that B-1 cells, mainly B-1b cells, proliferate in stationary cultures of normal mouse peritoneal cells and differentiate into a mononuclear phagocyte in vitro and in vivo.

These cells appear in cultures of normal adherent non-stimulated mouse peritoneal cells provided that the medium is not renewed for up to 7 days. This observation indicates that undetermined factor(s) are produced in these cultures which induce B-1 cell proliferation as demonstrated by [3H]thymidine incorporation. Borrello and Phipps (31) have already demonstrated that fibroblast-secreted macrophage colony stimulating factor is responsible for the generation of B-1a cells in vitro.

Although a more comprehensive investigation on the morphology of these cells must be performed, they are small cells as evaluated by flow cytometry, with sizes varying from 8 to 30 µm. Their capacity to multiply in vitro and radiosensitivity demonstrated by either the irradiation of the cultures or of the animals from which the peritoneal cells were obtained was the first indication that they were not monocyte-derived macrophages. It is largely confirmed in the literature that macrophages are radioresistant (32) and multiply in vitro only under the influence of conditioned medium (33). Further evidence is based on the fact that they did not express the calcium-binding protein MRP-14, a sensitive marker of cells of myelomonocytic origin (3436).

The simultaneous expression of IgM, B-220, CD5 and Mac-1 antigens on the surface of these cells, as demonstrated by flow cytometry, allows us to diagnose them as B-1 cells. The detection of IgM in the supernatant of the cultures not only confirmed that they were synthesizing but also secreting this Ig into the medium. Nevertheless, the high proportion of this cell population was composed by B-1b cells considering that only 5% of them expressed the CD5 marker. It is interesting to note that B-1 cells isolated from the spleen which transform into macrophage-like cells in co-cultures with fibroblasts are CD5+ cells, indicating their origin from B-1a cells (10).

Thus, our results clearly indicate that the great majority (95%) of cells that proliferated in stationary cultures of adherent peritoneal cells was composed by B-1b lymphocytes. Co-culture of lymphocytes with fibroblast (10) is reported as a pre-requisite to induce B-1 cell proliferation in vitro. In the model here described, it is conceivable that adherent macrophages or another undetermined cell type are the source of factors that maintain the proliferative state of these cells in culture. Further, it was not yet determined whether they have the capacity to self-proliferate or emerge from undifferentiated precursors. Recently, Vink et al. (19) demonstrated high expansion of B-1 cells in vivo, mainly the B-1b subset, in transgenic mice that constitutively express IL-9. Whether this cytokine is involved in B-1 cell proliferation in the model herein described remains to be investigated.

Additional experiments gave support for the conclusion that the small round cells that proliferate in culture are B-1 cells. It is well established that Xid mice, a lineage which lacks Burton's tyrosine kinase, has impaired production of B-1 lymphocytes (29). When peritoneal cells from Xid mice were cultured, B-1 lymphocytes were no longer observed, thus indicating that these mice are deprived not only of B-1 cells but also of their precursors.

Morphological results showed that B-1 cells remain either in suspension or adherent to the cell membrane of typically differentiated macrophages. Nevertheless, when they were collected and transferred to a new culture medium, a large proportion of them firmly adhered to the plastic surface, and spread to transform into bipolar and large mononuclear cells. It is interesting to note that not all small cells transform into large bipolar cells.

Considering that the non-adherent cell population collected from the 7-day cultures is heterogeneous, as demonstrated by flow cytometry analysis, the characteristics of the cells that generated the macrophage-like cells had to be determined. First, they were completely eliminated from the cell suspension when, before re-culturing, they were treated with an anti-IgM mAb plus complement, thus indicating that IgM is expressed on their outer membrane. Second, they co-express IgM and Mac-1 receptors when analyzed by flow cytometry. These data support the interpretation that these macrophage-like cells are derived from B-1 cells.

Our results and those obtained by Borello and Phipps (31) pose a basic question: should the B-1 cell-derived mononuclear phagocyte be considered a `macrophage'? Like monocyte-derived macrophages, they spread on the plastic surface and phagocytose via Fc and mannose receptors. Further, a proportion of them lose the ability to express IgM and B-220 on their surface, becoming only Mac-1+ cells, a phenotype that could not be distinguished from `classical' macrophages. As at the moment there is not enough information to prove that B-1 cell-derived mononuclear phagocytes share the same physiologic characteristics of monocyte-derived macrophages, we suggest they should be classified as a novel mononuclear phagocyte, the `B-1 cell-derived mononuclear phagocyte'. From our point of view, B-1 cells do not break the so-called `lineage fidelity' by expressing macrophage, B and T cell markers. Instead, the expression of these surface markers characterizes a distinct cell lineage with already known physiologic characteristics as proposed by Herzenberg (13).

Our data do not provide evidence that B-1a and B-1b cells transformation into a mononuclear phagocyte in vitro might occur in vivo. However, indirect evidence suggests that B-1 cells can exit from the peritoneal cavity and migrate to other tissues. It has been demonstrated that B-1 cells can be detected in periodontal lesions in man (37) and in delayed hypersensitivity-mediated lesions in mice (23). When B-1a cells were inoculated into the peritoneal cavity of SCID mice they were detected as IgA-secreting plasmocytes in the lamina propria of the intestine of the animals (24). These observations led us to investigate whether B-1 cells cultured in vitro, as here described, had the ability to migrate from the peritoneal cavity to a non-specific inflammatory milieu. Results clearly show that when B-1 cells were labeled in vitro with [3H]thymidine and injected into the peritoneal cavity of normal mice, labeled cells migrated to a non-specific inflammatory focus, adhered to the glass surface and differentiated into a macrophage-like cell. Further evidence that these cells migrate to a site of inflammation was the demonstration of macrophage-like cells on the coverslip surface expressing IgM epitopes. Although it was not demonstrated whether these cells derived from B-1a or B-1b cells and whether they were phagocytic or not, this finding suggests that the nature and origin of mononuclear cells which migrate to an inflammatory milieu as previously established (2,3,3840) should be revisited.

The role of these cells on the fate of the inflammatory response and on parasite resistance to infection was not evaluated. However, it has been clearly demonstrated that Xid mice are significantly more resistant to T. cruzi (41), Paracoccidioides brasiliensis (A. P. Kipinis, pers. commun.) and lymphatic filarial parasite (42) infections. These data suggest that B-1 cells might down-regulate the efficacy of effector cells, such as macrophages, to eliminate parasites in the inflammatory milieu. The fact that these cells are the main source of B cell derived IL-10 (43) supports this hypothesis.

It has also been demonstrated that B-1 cells are involved in the pathogenesis of autoimmune diseases (2426). In this direction, it has recently been reported that autoimmune manifestation is abrogated in Lyn-deficient mice by mutation of the Btk gene (22).

Undoubtedly, the demonstration that B-1 cells exit coelomatic cavities and migrate to an inflammatory site to transform into a novel type of mononuclear phagocyte, brings new insights for the investigation of the role these cells could play in inflammatory, degenerative and neoplasic pathologies.


    Acknowledgments
 
The authors are pleased to acknowledge financial support by the state-granting agency (FAPESP) and by the National Research Council (CNPq). We are also indebted to Professor Telma Zorn for the preparation of the histoautoradiograms and Professor Renato A. Mortara for his skilful orientation in the confocal analysis.


    Abbreviations
 
LPS lipopolysaccharide
SRBC sheep red blood cell

    Notes
 
Transmitting editor: C. Martinez-A

Received 3 May 2000, accepted 19 June 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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