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Originally published online as doi:10.1189/jlb.1004624 on July 21, 2005

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(Journal of Leukocyte Biology. 2005;78:845-852.)
© 2005 by Society for Leukocyte Biology

Macrophages in the murine pancreas and their involvement in fetal endocrine development in vitro

S. B. Geutskens*,1, T. Otonkoski{dagger}, M-A. Pulkkinen{dagger}, H. A. Drexhage* and P. J. M. Leenen*

* Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; and
{dagger} Hospital for Children and Adolescents and Biomedicum, Program of Developmental and Reproductive Biology, University of Helsinki, Finland

1Correspondence: Department of Molecular Cell Biology, Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail: s.geutskens{at}sanquin.nl


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ABSTRACT
 
Macrophages are a heterogeneous population of cells that belong to the mononuclear phagocyte system. They play an important role in tissue homeostasis and remodeling and are also potent immune regulators. Pancreatic macrophages are critically involved in the development and pathogenesis of autoimmune diabetes. To elucidate the ontogeny of pancreatic macrophages, we characterized in this study the macrophages present in the adult and developing fetal pancreas of normal mice. We additionally examined the presence of local macrophage precursors and the involvement of macrophages in the growth of endocrine tissue in the fetal pancreas. We identified two phenotypically distinct macrophage subsets in the adult pancreas. The majority of macrophages was CD45+ER-MP23+MOMA-1+. Under noninflammatory conditions, only a minority (~5%) of the pancreatic macrophages additionally expressed the macrophage marker F4/80. In contrast, in the fetal pancreas, phenotypically, mature macrophages were identified exclusively by their expression of F4/80 and lacked detectable staining with ER-MP23 and MOMA-1 antibodies. In fetal pancreas organ cultures, we could show that macrophages develop from pre-existing precursors, which are present in the fetal pancreas at embryonic age 12.5. Moreover, the number of macrophages increased significantly when macrophage-colony stimulating factor was added to these cultures. It is important that this increase of F4/80-positive cells was paralleled by an increase in the number of insulin-producing cells, suggesting that macrophages support the growth of these endocrine cells.

Key Words: embryo • macrophage precursor • M-CSF • insulin-producing cells


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INTRODUCTION
 
Macrophages belong to the mononuclear phagocyte system and regarding their function and phenotype, comprise a heterogeneous population of cells. Next to their important immune-regulatory role, they are also involved in the remodeling of various tissues during embryonic development and adult life [1 ]. Unfortunately, macrophage activity does not always have a beneficial outcome, and macrophages are believed to play key roles in the autoimmune process leading to type 1 diabetes [2 3 4 5 ]. In nonobese diabetic (NOD) mice, which develop diabetes spontaneously, macrophages are among the first leukocytes that accumulate around the insulin-producing islets before lymphocyte reactivity against islet cells is evident. The early intra-islet infiltration of macrophages is associated with the progression of insulitis and finally, the development of diabetes. Yet, the ontogeny and primary function of pancreatic macrophages under noninflammatory conditions are not known.

It is generally accepted that bone marrow (BM)-derived monocytes are the blood-borne precursors of macrophages [6 ], but the ontogeny of resident tissue macrophages overall is poorly understood. Under inflammatory conditions, monocytes will infiltrate affected peripheral tissues in response to acute or chronic stimuli and differentiate into dendritic cells (DCs) or exudate macrophages [7 ]. Murine peripheral blood monocytes express differential levels of Ly-6C, uniform levels of the early myeloid marker ER-MP58, high levels of CD11b, low levels of F4/80, and insignificant levels of markers specific for mature macrophages such as CD16/CD32 [8 , 9 ]. BM-derived monocytes rapidly lose the expression of Ly-6C upon differentiation into macrophages in vivo [10 ] or in vitro, when stimulated with macrophage-colony stimulating factor (M-CSF) [11 ].

It is not certain whether tissue macrophages are replenished by blood monocytes under noninflammatory conditions. Adult tissue-resident macrophage populations, which are maintained independently from circulating monocytes, have been identified in different organs, and they possibly derive from a BM-independent precursor pool that proliferates locally [12 ]. In conjunction, populations of resident macrophage subsets, such as Kupffer cells in the liver and microglia in the central nervous system, may derive from primary seedings of fetal macrophages [13 14 15 16 ].

In the early postnatal pancreas of various mouse strains, mature F4/80+ (BM8+), ER-MP23+, and MOMA-1+ macrophages [17 18 19 ] and CD11c+ DCs have been detected from birth on [3 , 20 ]. Their frequency and, in particular, that of the F4/80+ macrophages significantly decrease from birth to weaning, possibly as the result of exocrine expansion during the postnatal development of the pancreas.

In spite of the important role in diabetogenesis, which was suggested for pancreatic macrophages, they have not been fully characterized in the noninflamed pancreas. The distinctive tissue localization and age-related changes in the frequency of cells labeled positively for the different macrophage markers suggest that different macrophage subsets are present in the mouse pancreas. The examination of the pancreatic macrophage compartment under noninflammatory conditions may give more insight into their ontogeny and normal function.

Therefore, we characterized in this study the macrophages present in the adult and developing fetal pancreas under noninflammatory conditions by using different combinations of antibodies (see Table 1 ) against antigens expressed by mature resident macrophages such as F4/80, MOMA-1, ER-MP23, CD115 [M-CSF receptor (M-CSFR)], and CD11b and antibodies against macrophage precursors such as ER-MP12 (anti-CD31), ER-MP20 (anti-Ly-6C), and ER-MP58. In addition, we have examined the presence of early macrophage precursors in fetal pancreas by culturing tissue explants with or without M-CSF and analyzing the presence of macrophages thereafter. To investigate the role of pancreatic macrophages in endocrine tissue development, we have studied, in parallel, the development of insulin- and glucagon-producing cells in these cultured fetal pancreas explants.


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  		Table 1. Description of Antibodies Used for Immunohistochemistry/Immunofluorescence


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MATERIALS AND METHODS
 
Animals
Pregnant C57BL/6 female mice were bred at the animal facility of Biomedicum (Helsinki, Finland). Mothers were killed, and embryos were removed at embryonic ages (E)12.5, E14.5, and E17.5. Adult, 7-week-old, female C57BL/6 and BALB/c mice were obtained from Harlan (Horst, The Netherlands) and were kept at the animal facility of the Erasmus MC (Rotterdam, The Netherlands). Adult mice were killed at 11 or 35 weeks of age. All strains were kept under specific pathogen-free conditions and fed ad libitum. A minimum of five animals per age/strain/group was used for all studies. Animal use and handling followed the ethical guidelines provided by the European Union.

Pancreatic explant culture
Pancreatic explant cultures of C57BL/6 mice of E12.5 were used to study the presence of early macrophage precursors. The appearance of the vaginal plug was noted as E0.5. The embryonic duodenal loop along with the dorsal and ventral pancreatic bud and stomach were microdissected and cultured according to methods designed for embryonic kidney culture [21 , 22 ]. Briefly, the explants were placed on Nucleopore filters (1.0 µm pore size, Costar, Corning, NY) on metal grids and cultured at the air-liquid interphase in serum-free, improved modified Eagle’s medium (Life Technologies, Inc., Gaithersburg, MD), supplemented with transferrin (30 µg/ ml), penicillin (100 IU/ml), and streptomycin (100 µg/ml), with or without 50 ng/ml recombinant murine M-CSF (R&D Systems, Abingdon, UK). After 5 days of culture, the explants were embedded in Tissue-Tek (Miles, Elkart, IN), frozen in dry ice, chilled isopentane, and stored at –80°C or fixed for 4 h at room temperature (RT) in Bouin’s fixative, rinsed with 50% alcohol, and stored in 70% alcohol before dehydration and paraffin embedding.

Immunohistochemistry and immunofluorescence
Adult pancreases were embedded in Tissue-Tek (Miles) and frozen in liquid nitrogen. Cryostat sections (5 µm-thick) were cut at 100–200 µm intervals in a series of five or six sections per pancreas, dried, and stored at –80°C. Fetal pancreases or cultured explants were cross-sectioned entirely in 5 µm-thick cryostat sections or 2 µm-thick paraffin sections. Cryosections were fixed for 10 min in acetone containing 0.03% hydrogen peroxide and subsequently air-dried. Paraffin-embedded sections were deparaffinized, fixed for 30 min in methanol containing 0.03% hydrogen peroxide, and rehydrated. Further processing followed similar proceedings as the cryosections.

Sections were incubated with 2% normal mouse serum (NMS) in phosphate-buffered saline (PBS) for 1 h at RT to block nonspecific binding sites. If stainings were amplified with Tyramide Signal AmplificationTM, NMS was diluted in 0.1 M Tris-Cl, 0.15 M NaCl, and 0.5% blocking reagent (Perkin Elmer Life Sciences, Boston, MA). If biotinylated antibodies were used, an additional blocking step against endogenous avidin and biotin was applied according to the manufacturer’s protocol (Vector Laboratories, Burlingame, CA). Slides were washed in PBS with 0.05% Tween-20 and incubated overnight at 4°C with primary antibodies specific for different macrophage or endothelial or endocrine markers (Table 1 ). Sections were washed with PBS-Tween-20 and incubated with appropriate secondary antibodies in the presence of 1.5% NMS [peroxidase-conjugated: swine anti-guinea pig (PO141), swine anti-rabbit peroxidase (PO399), and goat anti-rat (PO162, all Dako); alkaline phosphatase-conjugated: swine anti-rabbit (DO306; Dako); or fluorochrome-conjugated: F(ab')2 goat anti-rat fluorescein isothiocyanate (FITC; Serotec GmbH, Düsseldorf, Germany] and goat anti-rabbit tetramethylrhodamine isothiocyanate (BI 2207, Biosys S.A., Compiègne, France). Biotin-conjugated antibodies were detected with streptavidin Texas red (Caltag). The 3-amino-9-ethylcarbazole substrate (Sigma Chemical Co., St. Louis, MO) was used for detecting specific peroxidase activity in 50 mM sodium acetate/0.02% hydrogen peroxide. Fast-blue BB base was used to detect alkaline phosphatase activity.

Immunohistochemical stainings were counterstained with Mayers hemalum solution (Merck, Darmstadt, Germany) and embedded in Kaisers glycerol gelatin (Merck). Immunofluorescence stainings were embedded in 4',6'-diamidino-2-phenylindole-containing Vectashield (Vector Laboratories). The resulting labeling was examined by normal light microscopy or fluorescence microscopy. Adult spleen and liver served as positive control tissue for all stainings.

Cell quantification
To quantify macrophages and insulin- and glucagon-expressing cell numbers after explant culture, every fourth section of the cross-sectioned explants was successively stained for F4/80 (cryostat sections), insulin, or glucagon (paraffin sections). For each antibody, a minimum of 16 sections per explant, each section cut at a different level, was stained. Positively labeled cells with visible nuclei were counted by light microscopy at an optical magnification of 400x. The corresponding surfaces of the pancreas explants were measured by marking the border with a computer-interfaced freehand tool at an optical magnification of 8x. Data were analyzed with the VIDAS-RT software (Kontron Elektronik GmbH/Carl Zeiss, Weesp, The Netherlands).

Statistical analysis
Data were expressed as mean ± SD; P values were calculated with the Mann-Whitney U-test.


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RESULTS
 
ER-MP23+ F4/80– macrophages represent the largest resident population in the adult pancreas
High numbers of ER-MP23+ macrophages were found randomly scattered in the connective tissue of inter- and intralobular septa and perivascular areas of the adult pancreas (Fig. 1A ). A similar distribution pattern was observed for the labeling with MOMA-1 antibody (Fig. 1B) , a marker expressed by metallophilic macrophages in the spleen and lymph nodes, and by Kupffer cells in the liver. As compared with the number of ER-MP23+ cells, cells positive for the M-CSFR CD115 (Fig. 1C) , the general macrophage marker F4/80 (Fig. 1D) , or the ß2 integrins CD11b (Fig. 1E) or CD11c (not shown) were rarely observed (Table 2 ) and were only occasionally found in septa or the outer pancreatic capsule.



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  		Figure 1. Differential labeling patterns of markers for mature macrophages and macrophage precursors in the adult murine pancreas. Antibodies recognizing antigens expressed on mature macrophages or macrophage precursors showed a differential labeling pattern in the pancreas, here shown for C57BL/6 mice at 9 weeks of age. Antibodies used are depicted in the figure. Symbols depicted indicate: i, Islet; d, duct; v, vascular structures; original magnifications, 200x.


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  		Table 2. Number of Macrophages in the Adult Murine Pancreas Relative to the Total Pancreas Surface Area (mm2)

Different distribution patterns were observed for antigens expressed by macrophage precursors. Cells positively labeled with the myeloid precursor-specific antibody ER-MP58 were observed (Fig. 1F) , although rarely, whereas individual cells expressing the myeloid precursor markers CD31 (recognized by ER-MP12; Fig. 1G ) and Ly-6C (recognized by ER-MP20; Fig. 1H ) were expressed widely throughout the exocrine tissue. These latter two antibodies additionally labeled vascular structures in the pancreas.

To assess the hematopoietic origin of the myeloid marker-expressing cells, we used fluorescent double-labeling with the pan-leukocyte marker CD45. Labeling for CD45 was observed on all ER-MP23+ cells as well as on all F4/80+ and CD11b+ cells (not shown). The ER-MP23+ cells were also labeled by the MOMA-1 antibody (Fig. 2A ). Labeling with F4/80 was observed on a restricted number of ER-MP23+ cells, as is consistent with the lower number of F4/80+ cells present in the adult pancreas (Fig. 2B) . CD115 labeling was detected on few ER-MP23+ cells only (Fig. 2C) .



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  		Figure 2. The adult pancreatic macrophage compartment is comprised of phenotypically distinct, mature macrophage subsets. Immunofluorescent labeling of frozen pancreas sections with combinations of different antibodies recognizing antigens expressed by mature macrophages (A–C) or macrophage precursors (D–H). Conjugates used: Texas red (red labeling) combined with FITC (green labeling). (A–F) Antibodies used are depicted. Colocalization appears as yellow labeling in the merged panels on the right. (G) CD45-positive cells (green) do not colocalize with CD31-positive cells (red). (H) Vascular structures are labeled with the endothelial marker von Willebrand factor (VWF; red labeling) and ER-MP20 (Ly-6C, green labeling). Symbols depicted indicate: i, Islet; d, duct; v, vascular structures. Original magnifications: (A and E) 1000x; (B, D, and F) 600x; (C, G, and H) 160x.

Labeling for ER-MP58 colocalized with CD45 (Fig. 2D) but not with that of Ly-6C (Fig. 2E) . Most, if not all, Ly-6C+ cells did express CD31 (Fig. 2F) . Notably, neither labeling with CD31 (Fig. 2G) nor with Ly-6C antibodies (not shown) colocalized with that of CD45. CD31 and Ly-6C colocalized with the endothelial marker VWF on endothelial cells of the vascular structures of the pancreas (Fig. 2H) .

Mature CD45+F4/80+ macrophages are first observed at E14.5 in the fetal pancreas
By using immunofluorescence on fetal pancreas sections, CD45+ cells were readily detected in the pancreatic buds, duodenal loop, and stomach at E12.5 (Fig. 3A ), the earliest embryonic age examined. However, labeling for markers of mature macrophages was not observed at this age. CD45+F4/80+ cells were first observed in the pancreas at E14.5 (Fig. 3B) in parallel with the appearance of F4/80-, CD11b-, and CD115-expressing cells in the fetal liver at E14.5 (not shown). Labeling for macrophage markers other than F4/80, such as CD11b, CD115, ER-MP23, and MOMA-1, was not detected in the pancreas at E14.5. CD45+F4/80+ macrophages were still present in the fetal pancreas at E17.5 (Fig. 3C) , and some macrophages expressed CD11b at this age as well (Fig. 3D) . CD115+ cells were observed in a comparable number and distribution as F4/80+ cells at E17.5 (Table 3 ), whereas few ER-MP23+ cells were present in the embryonic pancreas from E17.5 onward (not shown).



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  		Figure 3. CD45+ F4/80+ mature macrophages are present in the fetal pancreas. Immunofluorescent labeling of frozen fetal pancreas sections with combinations of different antibodies recognizing antigens expressed by mature macrophages. Conjugates used: Texas red (red labeling) combined with FITC (green labeling). Antibodies used are indicated. Colocalization appears as yellow labeling in the merged panels on the right. (A) Pancreatic bud of C57BL/6 mouse at E12.5. (B and E) Pancreas of C57BL/6 mouse at E14.5. (C, D, and F) Pancreas of C57BL/6 mouse at E17.5. Original magnifications: (A) 400x; (B–D) 600x; (E and F) 160x; right panels show a higher original magnification (400x) of the selected sections indicated in the merged image of the F4/80+ and insulin double-staining. The arrows indicate islet-associated cells counted.


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  		Table 3. Percentage of Macrophages Associated to Insulin+ Islets in the Fetal C57BL/6 Pancreas

Expression of MOMA-1 was not observed at any age, in any of the fetal pancreases examined. It is interesting that F4/80+ cells were frequently found in close apposition to insulin-producing cells at E14.5 and more clearly at E17.5 (Fig. 3E and 3F) . Quantification of the total number of F4/80+ cells or CD115+ cells in cross-sectioned fetal pancreases simultaneously stained for insulin showed that over 20% of F4/80+ cells associated with insulin+ islets at E14.5. At E17.5, this percentage increased twofold, and up to 45% of F4/80+ cells associated with islets at this age (Table 3 and Fig . 3E and 3F ; arrows indicate islet-associated cells counted).

Cells positively labeled for the early myeloid precursor markers CD31, Ly-6C, and ER-MP58 were present in the fetal pancreas from E12.5 onward (data not shown). In the fetal liver, CD31 and Ly-6C were expressed by CD45+ and CD45– cells at E14.5 (not shown). However, the labeling of CD31 and Ly-6C in the fetal pancreas did not colocalize with CD45, suggesting that these markers were expressed by endothelial structures similar to what had been observed for the adult pancreas. ER-MP58+ cells did express CD45 in the fetal pancreas at E17.5, although labeling for ER-MP58 was rarely observed (not shown).

Macrophage precursors are present in the fetal pancreas at E12.5
To examine the presence of macrophage precursors before any labeling of mature macrophage markers was detected in the fetal pancreas, pancreatic buds along with the stomach and duodenal loops were excised at E12.5 and cultured for 5 days with or without M-CSF. It is interesting that after 5 days of culture, F4/80+ cells were indeed observed in the explants, even without the addition of exogenous M-CSF (Fig. 4A ). In accordance with the labeling of fetal pancreases at E17.5, CD11b was the only other macrophage marker observed in the cultured explants (not shown). Labeling of other mature macrophage markers was not detected, not even after immunohistochemical amplification of the signal. Thus, intrapancreatic precursors were present at E12.5 and capable to differentiate into mature macrophages, independent of the addition of M-CSF. However, supplying M-CSF induced a threefold increase in the number of F4/80+ cells present per total explant surface area (Fig. 4B and 4C) .



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  		Figure 4. Exogenous M-CSF significantly increases the number of F4/80+ cells in cultured E12.5 pancreas explants. F4/80-positive cells are readily observed in pancreas explants cultured for 5 days without M-CSF (A) or with M-CSF (B). Original magnification, 200x. The number of F4/80-positive cells relative to the total explant surface area/mm2 ± SD is depicted for explants cultured without M-CSF (Control; n=5) or with M-CSF (n=5).

Exogenous M-CSF affects endocrine cell growth in fetal explants in vitro
As macrophages are involved in tissue development, we investigated whether the addition of M-CSF, resulting in increased macrophage numbers, affected the development of insulin- and glucagon-expressing cells in the cultured explants. Insulin-producing cells were readily detected in explants cultured with M-CSF (Fig. 5A and 5B ) or without M-CSF (not shown), similar to glucagon-producing cells (Fig. 5A and 5B) , which were found adjacent to the insulin-producing cells. It is interesting that the number of insulin+ cells significantly increased more than fourfold when explants were cultured with M-CSF. An increase in the number of glucagon+ cells was also observed but to a much lower extent (Fig. 5C) , and the difference did not reach statistical significance.



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  		Figure 5. Pancreas explants cultured with M-CSF have increased numbers of insulin-producing cells. (A) Insulin (red)- and glucagon (blue)-producing cells are observed in the explants after 5 days of culture. Symbols depicted indicate: s, Lumen of stomach; i, intestine; e, endocrine tissue. Original magnifications, 40x and 200x, respectively. Sections were counterstained with hematoxylin. The number of insulin (B)- and glucagon (C)-producing cells relative to the total explant surface area/mm2 ± SD is depicted for explants cultured without M-CSF (Control; n=8) or with M-CSF (n=5).


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DISCUSSION
 
By using different combinations of antibodies recognizing antigens expressed by mature macrophages or macrophage precursors, we have determined the phenotype of resident macrophages in the noninflamed adult pancreas as CD45+ER-MP23+MOMA-1+ cells. These macrophages are distributed randomly in the connective tissue of the adult pancreas in high numbers. It is interesting that in the adult noninflamed pancreas, only a small subset of these cells is positive for the F4/80 antigen, an antigen that is regarded as a general marker for macrophages. By contrast, in the NOD pancreas, F4/80+ cells are abundantly present, even prior to the peri-islet accumulation of T-lymphocytes [3 ]. Their presence is correlated to islet destruction, and they are virtually absent in BDC2.5 T cell receptor-transgenic NOD mice, which have a strongly reduced incidence of diabetes [23 ]. This suggests that the pancreatic F4/80+ cells in the NOD mouse possibly represent a subset of inflammatory macrophages, which is distinct from the resident CD45+ER-MP23+MOMA-1+ macrophages in the noninflamed pancreas. They may have been recruited to the pancreas recently or gained expression of F4/80 to detectable levels in response to local inflammatory stimuli.

Phenotypically mature macrophages were not observed in the fetal pancreas until E14.5, although CD45+ cells were already present in the pancreatic buds and surrounding tissues from E12.5 on. In contrast to the pancreatic macrophages of adult mice, mature macrophages in fetal pancreases were all characterized by the expression of F4/80, and at later stages, a subpopulation of these cells was shown to express CD11b additionally. The F4/80+ cells, which were observed at E14.5, do not likely enter the pancreas via the blood circulation, as functional blood vessels containing a lumen are not observed until E15.5 in the murine pancreas [24 ]. ER-MP23+ cells were not observed until E17.5, and other markers such as MOMA-1 or CD11c were not observed at all before birth. The order of appearance of the expression of these different macrophage markers in the pancreas followed the kinetics of appearance that was described for other embryonic tissues. F4/80 was the first marker observed in the liver, spleen, thymus, peritoneum, and kidney from E12 on [25 ], followed by the expression of CD11b in most of these tissues [26 ].

To establish the presence of macrophage precursors, we cultured fetal pancreatic buds that were excised at the age of E12.5. At this stage, the pancreatic buds contain CD45+ cells exclusively. It is interesting that F4/80+CD11b+ cells were observed when the excised E12.5 pancreatic anlage was further cultured for 5 days in serum-free medium. This indicates the presence of precursors at E12.5, which possesses the capacity to differentiate into cells with a macrophage-like F4/80+CD11b+ phenotype.

Unfortunately, we were not able to establish the phenotype of the early macrophage precursors unequivocally. In the adult and fetal pancreas, the myeloid precursor markers CD31 and Ly-6C were expressed clearly by vascular structures and by single cells, which lacked the expression of CD45. The ER-MP58 antibody labeled individually scattered cells expressing CD45. However, labeling with ER-MP58 is not fully specific for macrophage precursors, as ER-MP58 additionally recognizes granulocytes and their precursors [27 ].

In accordance with the labeling observed in the fetal pancreas in vivo, the expression of ER-MP23 or other mature macrophage markers was not observed in the cultured fetal pancreas explants. This may suggest that ER-MP23+MOMA-1+ cells represent a distinct population of macrophages, which is seeded at later stages of gestation, possibly via the vasculature, and their presence appears to be continued after birth. A rapid decline in the number of F4/80+ cells was observed in the pancreas from birth to weaning [20 ]. This decline may be explained by the expansion of the exocrine pancreas during this period but can also be suggestive for the disappearance of the earliest F4/80+ fetal macrophages. Alternatively, the expression of F4/80 may be down-regulated during the retention of macrophages in the pancreas paralleled by the acquisition of ER-MP23 and MOMA-1 expression. Unfortunately, it is presently not possible to label the F4/80+ cells in the fetal pancreas in vivo to track their differentiation and localization during the development to adulthood. F4/80 expression normally increases with cell maturation [28 ], and migrating Langerhans cells have been reported to lose the expression of F4/80 upon their maturation into lymph node DCs [29 ]. However, the mechanisms that regulate the expression of F4/80, ER-MP23, and MOMA-1 are still unknown. The current phenotypic analysis, therefore, merely allows speculation about the developmental relationship of the F4/80+ fetal macrophages and F4/80+ and F4/80– macrophages observed in the adult pancreas. It is interesting that the presence of F4/80+ macrophages is observed during periods of extensive tissue turnover in the normal fetal mouse pancreas as well as in the pancreas of adult NOD mice. The presence of F4/80+ macrophages has been correlated to tissue organogenesis before [30 ]. Moreover, results described in a recent paper by Banaei-Bouchareb et al. [31 ] indicate that the presence of pancreatic macrophages is important for normal islet development in op/op mice in vivo, and their data are supported by our data in vitro.

In the cultured fetal pancreas explants, the precursors differentiated without the addition of exogenous M-CSF, possibly induced by endogenous M-CSF or other growth factors present within the tissue during culture. It is important that we showed that the addition of M-CSF to the cultures resulted in a threefold increase in the number of F4/80+ cells. The presence of M-CSF-dependent and -independent macrophage populations was shown recently in the pancreas of op/op mice [31 ]. These mice lack functional M-CSF and are devoid of several macrophage populations in different organs. F4/80+ cells were detected in the pancreas of op/op mice at E18.5 (albeit at low numbers), supporting the presence of two distinct pancreatic macrophage populations. It is interesting that the macrophage-deficient op/op mice show a severe decrease in insulin mass as a result of hypoplasia and decreased proliferation of insulin+ cells. In accordance with those results, we show here that the addition of M-CSF to the fetal explant cultures resulted in a fourfold increase of the number of insulin+ cells in vitro. The effect of M-CSF is likely mediated via the differentiation and activation of the macrophage precursors that were present in the explant. Double-labeling studies indicate that indeed, a significant proportion of macrophages in the fetal pancreas associates with developing islets. M-CSF increases the expression and production of interleukin (IL)-6 in macrophages [32 ], and enhanced expression of IL-6 results in islet hyperplasia, neo-ductular formation [33 ], and increased expression of preproinsulin mRNA [34 ]. IL-6 can thus be involved in islet metabolism as well as islet neogenesis.

To conclude, we show here that the resident pancreatic macrophage compartment in adult mice is not represented by a single phenotype but comprises different phenotypes with a major population of ER-MP23+MOMA-1+F4/80–CD45+ cells and a minor population of ER-MP23+MOMA-1+F4/80+CD45+ cells. Fetal pancreatic macrophages are identified exclusively by the expression of F4/80, and they appear to play an important role in the growth of endocrine tissue in the fetal pancreas in vitro.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the European Committee (QLRT-1999-00276-"MONODIAB") and Stichting Termeulenfonds, The Netherlands. The authors thank Ms. P. Kinnunen for excellent technical assistance and Dr. J. J. Bajramovic for critically reading this manuscript.

Received October 30, 2004; revised April 20, 2005; accepted May 24, 2005.


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