Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0903442 on January 14, 2004

Published online before print January 14, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0903442v1
75/4/612    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Burnett, S. H.
Right arrow Articles by Cohen, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Burnett, S. H.
Right arrow Articles by Cohen, D. A.
(Journal of Leukocyte Biology. 2004;75:612-623.)
© 2004 by Society for Leukocyte Biology

Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene

Sandra H. Burnett, Edward J. Kershen, Jiayou Zhang, Li Zeng, Susan C. Straley, Alan M. Kaplan and Donald A. Cohen1

Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington

1Correspondence: Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Room MS419, Lexington, Kentucky 40536-0084. E-mail: dcohen{at}pop.uky.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice expressing an inducible suicide gene, which allows systemic and reversible elimination of macrophages, were developed. A macrophage-specific c-fms promoter was used to express enhanced green fluorescent protein and a drug-inducible suicide gene that leads to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage. Transgenic mice were fertile, of normal weight, and showed no abnormal phenotype before drug exposure. The transgene was expressed constitutively in macrophages and dendritic cells (DC) but not significantly in T cells or B cells. Induction of the suicide gene led to depletion of 70–95% of macrophages and DC in nearly all tissues examined. Depletion reduced the ability to clear bacteria from the blood and led to increased bacterial growth in the liver. Depleted mice displayed several abnormalities, including splenomegaly, lymphadenopathy, thymic atrophy, extramedullary hematopoiesis, and development of peritoneal adhesions. This new, transgenic line will be useful in investigating the role of macrophages and DC.

Key Words: depletion • c-fms • dendritic cell • GFP • peritoneal adhesion • Yersinia pestis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages and other antigen presenting cells (APC) are essential for the development of innate and adaptive immunity by virtue of their microbicidal activity and their ability to phagocytize, process, and present antigen to naive T cells. Studies in which macrophages have been eliminated in vivo by using depleting agents such as silica, carageenen, gadolinium chloride, and chlodronate [1 2 3 4 ] have elucidated a number of important immune functions of macrophages. Although these reagents have been useful over the years, their use for in vivo studies has been limited in that these approaches generally do not result in systemic elimination of macrophages. Gene knockout approaches have the potential to remove macrophages systemically to study their functions in vivo in a mature immune system. However, the importance of these cell types in embryonic development has hampered the use of gene knockout mice for studies of the mature immune system [5 , 6 ]. Conditional knockout of genes that block macrophage maturation, such as can be generated with a cre-lox system [7 ], have other technical problems such as a slow turnover of mature macrophages already present in tissues at the time of gene knockout induction. In contrast, an inducible suicide gene that functions independent of the cell cycle and is expressed in a macrophage-specific manner would overcome all of the above problems. Embryonic development would occur in the presence of a normal number of macrophages, and an appropriate suicide gene could rapidly eliminate macrophages systemically upon activation of the suicide mechanism. Moreover, systemic macrophage ablation would be transient and reversible upon removal of the suicide activator, provided the suicide gene was not expressed in stem cells.

Several promoters could be used to drive expression of a suicide gene in a macrophage-specific manner such as the PU.1 transcription factor [5 , 6 ], macrosialin (CD68) [8 ], and macrophage colony-stimulating factor (CSF)-1 receptor (c-fms) [9 , 10 ]. For construction of transgenic mice, we selected the c-fms promoter, which is known to promote expression of the CSF-1 receptor predominantly on cells of the mononuclear phagocyte system in the mouse, including monocytes, macrophages, dendritic cells (DC), Kuppfer cells, Langerhans cells, osteoclasts, and microglial cells [11 ]. Intron 2 of the c-fms promoter is required for macrophage-specific expression [10 ]. In recent studies [12 ], expression of an enhanced green fluorescent protein (EGFP) reporter gene driven by a 7.2-kb murine c-fms promoter fragment inclusive of intron 2 and EGFP was detected in cells of the macrophage lineage in all tissues analyzed.

The herpes simplex virus thymidine kinase (HSVtk) gene has been used as a suicide gene in transgenic mice for in vivo depletion of thymocytes [13 ], T cells [14 ], B cells [15 ], and DC [16 ]. Upon activation by HSVtk, the antiviral gancyclovir is converted into a metabolite, resulting in cellular death of mitotically active HSVtk-expressing cells. HSVtk is less than ideal for systemic depletion of macrophages, as HSVtk does not cause cell death in resting cells. Conversely, a system capable of activating proteins in an apoptosis cascade can deplete cycling and noncycling cells.

Cross-linking the human Fas receptor (CD95, APO-1) results in activation of the caspase-8 pathway, which ultimately leads to apoptosis [17 ]. Spencer et al. [18 ] previously described construction of a suicide gene consisting of the cytoplasmic domain of Fas linked to two copies of the FK506-binding protein (FKBP) and a myristolation-targeting peptide for in vivo depletion of T cells. The cytoplasmic Fas was membrane-tethered by the myristolation-targeting peptide, and FK1012, a covalently linked dimer of FK506, induced Fas-mediated apoptosis by dimerization of proximal transgene proteins. Another similar construct was developed with the FKBP–Fas segment but replaced the myristolation region with the human low-affinity nerve growth factor receptor ({Delta}LNGFR) for membrane insertion and to serve as an otherwise nonfunctional epitope tag [19 ]. Ariad Pharmaceuticals (Cambridge, MA) developed AP20187 as a modified, improved form of FK1012 for dimerization of the suicide protein to activate the cytoplasmic Fas fragments and induce apoptosis in trangene-expressing cells without affecting endogenous FKBP.

Using genes for the macrophage-specific c-fms promoter EGFP and the {Delta}LNGFR–FKBP–Fas suicide construct, we developed transgenic macrophage Fas-induced apoptosis (Mafia) mice on a C57Bl/6J background in which conditional macrophage ablation can be induced. Bicistronic expression of EGFP and the suicide gene allows for identification of transgene-expressing cells in all tissues by EGFP expression and systemic depletion of macrophage-related cells by exposure to the AP20187 dimerizer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
All wild-type (WT) C57Bl/6J mice were purchased from Charles River Laboratories (Boston, MA). Pups derived from transgene-injected eggs and those that tested positive for the Mafia transgene were bred with WT C57Bl/6J mice to establish separate founder colonies. Mafia mice offspring from all founder lines were screened by polymerase chain reaction (PCR) on tail-snip DNA taken at weaning. All data presented in this manuscript were obtained from the offspring of the female founder line #2. EGFP expression was confirmed by flow cytometric analysis of peritoneal cells collected by lavage with phosphate-buffered saline (PBS) after anesthetization with 2.5% avertin. Negative mice were eliminated from the colony. The University of Kentucky Division of Laboratory Animal Resources (Lexington) maintained all animals according to the guidelines of the Animal Welfare Act.

Transgene construction
The Fas-inducing segment of the transgene was kindly provided by Ariad Pharmaceuticals and contained the modified {Delta}LNGFR linked directly to two copies of the human FKBP and the cytoplasmic portion of human Fas protein. An internal ribosome entry site (IRES) sequence was inserted at the start codon of {Delta}LNGFR using NcoI. Dr. David Hume (University of Queensland, Australia) kindly provided the modified pGL-2 plasmid containing EGFP driven by the c-fms promoter [12 ]. The entire 11.1-kb Mafia transgene was generated by inserting a cassette containing IRES–{Delta}LNGFR–FKBP–FKBP–Fas into the NotI site immediately downstream from EGFP in the pGL-2 plasmid containing the c-fms promotor. The final plasmid containing the Mafia transgene was submitted to ACGT, Inc. (Northbrook, IL) for sequencing of overlapping regions at all ligation sites. The transgene (Fig. 1A ) was excised from the plasmid by FspI and partial SalI digestion and injected into pronuclei of fertilized eggs from superovulated C57Bl/6J mice, which had been mated with C57Bl/6J males. The University of Kentucky Transgenic Mouse Core Facility performed egg harvesting, microinjection, and implantation.



View larger version (46K):
[in this window]
[in a new window]
  		Figure 1. Elements of the Mafia transgene and coexpression of EGFP and the suicide protein. (A) Schematic representation of the Mafia transgene. Elements include: c-fms= 7.2 kb macrophage-specific promotor for CSF-1 receptor including intron 2; EGFP; IRES; {Delta}LNGFR; FKBP; Fas, human cytoplasmic domain of Fas; SV40 poly A, SV40 early mRNA polyadenylylation signal. The transgene product functions when exposed to the synthetic dimerizer, AP20187, which binds FKBP subunits of neighboring suicide proteins for dimerization and activation of the Fas subunits to initiate apoptosis. (B) Peritoneal cells from a WT and a Mafia mouse were stained with {Delta}LNGFR–APC and analyzed by flow cytometry.

 
Tissue culture
Cells collected by peritoneal lavage were counted and seeded for adherent cultures in 12-well, plastic-culture plates or for nonadherent cultures in Teflon inserts of 12-well plates (Savillex, Minnetonka, MN). Cells were grown in the presence of 5% CO2 in RPMI-1640 medium supplemented with glutamine, 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml; complete medium). For some of the initial in vitro depletion studies, peritoneal cells were cultured with 5 ng/ml interleukin (IL)-10, which helped to maintain high EGFP expression during in vitro culture as adherent cells [20 ]. Adherent peritoneal cell cultures were incubated in the presence or absence of 10 nM AP20187 for 24 h and were harvested by scraping. Cells were subsequently analyzed by flow cytometry to evaluate changes in the percentage of EGFP+ cells. For determination of the inhibitory concentration (IC)50, peritoneal cells were cultured in Teflon wells, as it was discovered that IL-10 was not necessary to maintain EGFP expression when culturing cells in suspension.

Dimerizer
AP20187 was a gift from Ariad Pharmaceuticals. Lyophilized AP20187 was dissolved in 100% ethanol at a concentration of 13.75 mg/ml or as a 1-mM stock solution in ethanol and stored at −20°C. For in vitro use, the 1-mM ethanol stock was diluted in complete culture medium to the desired concentration immediately before use. The final concentration of ethanol in the culture medium was below 0.5%. For in vivo use, peritoneal injections were prepared from the 13.75 mg/ml ethanol stock diluted to 0.55 mg/ml in an injection solution consisting of 4% ethanol, 10% PEG-400, and 1.7% Tween in water. All injections were administered to mice within 30 min of dilution into the injection solution. The volume of injection solution was adjusted according to mouse body weight to deliver 10 mg AP20187 per kg mouse. The average injection volume was 300 µl per mouse. Depletion data represent nine mice per tissue (combined from three independent studies) for 24 h data and seven mice per tissue (combined from two independent studies) for 7-day rest data.

Tissue collection
Animals were killed by CO2 asphyxiation. For single-cell preparations, fragments of liver, spleen, thymus, and brain were homogenized in 2 ml complete medium in a stomacher tissue homogenizer and then washed in complete medium or PBS before culture or analysis, respectively. Blood was obtained by cardiac puncture immediately following euthanasia. Lung tissue was digested in complete medium containing 1 µg/ml type I DNase and 20 units/ml collagenase for 45 min at 37°C before homogenization. Bone marrow was homogenized by passing marrow aspirates through a 26-gauge needle. Peritoneal lavage in killed mice was performed with 5 ml PBS. Cell preparations that contained blood were pelleted, resuspended in Tris-buffered ammonium chloride for 2 min, rinsed with 1% bovine serum albumin, 0.1% azide in PBS, filtered through a 70-µ pore-size filter, and rinsed again before staining for flow cytometric analysis. For immunohistological evaluation, fragments of liver, spleen, and lung were placed in histology cassettes and submerged in 4% paraformaldehyde overnight before embedding and staining.

Flow cytometry and cytospin
Flow cytometric analysis and sorting were performed on a MoFlo cell sorter (Cytomation, Fort Collins, CO), and collected data were analyzed using Cytomation Summit software. APC-labeled anti-{Delta}LNGFR antibody was obtained from Chromaprobe (Aptos, CA). Retinal pigment epithelial-Cy5-labeled F4/80 antibody was obtained from Serotec (Raleigh, NC). All other antibodies were obtained from BD PharMingen (Torrey Pines, CA). Cells were incubated on ice with anti-CD16/32 Fc{gamma}III/II receptor antibody for 10 min and then with an appropriate saturating concentration of labeled antibody for an additional 15 min. Cells were rinsed once in PBS before analysis. Depletion data for EGFP+ cells are based on data from regions, including only the EGFPhigh population. Sorted cells were centrifuged onto slides in a Cytospin3 (Shandon, Pittsburgh, PA) and stained with DiffQuick (Baxter Healthcare, Miami, FL).

Epidermal sheet preparation
EGFP+ Langerhans cells were visualized in wet mounts of freshly isolated epidermal sheets. Hair was removed from ears of killed mice with the use of a commercial depilation solution. Dissected ears were incubated for 2 h at 37ºC in PBS containing 1 µg/ml type I DNase and 20 units/ml collagenase. Epidermal sheets were then manually separated from the dermal layer and mounted in glycerol under a coverslip for immediate evaluation in a Zeiss Axiophot epifluorescent microscope.

PCR
DNA was extracted from tail snips by proteinase K (100 µg/ml) digestion at 56°C overnight. The Fast Start DNA Master SYBR Green I kit (Roche Molecular Biochemicals, Indianapolis, IN) was used for real-time PCR in a Roche LightCycler. Primers were custom-synthesized by Integrated DNA Technologies (Coralville, IA). Primer sequences for EGFP were 5' AGCTGACCCTGAAGTTCATCTG 3' forward and 5' ACGTTGTGGCTGTTGTAGTTGT 3' reverse; ß-actin primer sequences were intron-spanning 5' GTGGGCCGCTCTAGGCACCA 3' forward and 5' CGGTTGGCCTTAGGGTTCAGGGGGG 3' reverse. PCR was performed for 45 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 10 s. Linearized plasmid DNA containing the Mafia transgene was used as a positive control for EGFP and as a negative control for ß-actin. PCR for EGFP and ß-actin was performed on DNA samples from pups derived from the founder line to identify EGFP+ mice.

Immunohistochemistry (IHC)
Paraformaldehyde-fixed tissues were fixed, embedded, sectioned, and stained according to protocols for Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and the Antigen Retrieval Citra Solution (BioGenex, San Ramon, CA). As a result of the high-background fluorescence and an observed quench in EGFP fluorescence in formalin-fixed and paraffin-imbedded tissues, samples were stained with anti-GFP rather than directly observed for EGFP fluorescence. The anti-GFP–biotin (Vector Laboratories) primary antibody was used at a 1:200 dilution.

Bacterial infection study
Mice were treated 5 days with 10 mg/kg AP20187 or a mock-injection solution. Twenty-four hours after the fifth injection, mice were anesthetized with isoflurane and retro-orbitally injected with 100 µl PBS containing 5 x 104 {Delta}pgm Yersinia pestis KIM5 [21 ]. This strain of Y. pestis is conditionally virulent in that it is avirulent via a peripheral route of infection but fully virulent by the intravenous (i.v.) route. The bacteria were cultured at 26°C in heart infusion broth (BD Biosciences, Franklin Kales, NJ) for at least seven generations in exponential phase before use. They were washed once in PBS and resuspended in PBS to the desired density based on A620 (where an A620 of 1 equals 6x108 cells/ml). The actual dose given to the mice was determined by plating for viable numbers [colony-forming units (CFU)] on tryptose blood agar (BD Biosciences). For blood clearance studies, mice were killed 15 min postinfection, and 50 µl blood was diluted into 450 µl dH2O, spread onto nutrient agar plates, and cultured for 48 h before colony counting. For bacterial growth studies, infected mice were killed at 15 min and 3, 6, 9, and 24 h after infection. Livers were homogenized in water and plated on nutrient agar plates for colony enumeration. Four mice/treatment group were evaluated for each time point. Both studies were repeated to confirm data presented.

Statistical methods
Depletion data were first analyzed by ANOVA with tests for multiple comparisons. Data presented for flow cytometric and bacterial growth data are the result of Student’s t-tests on raw percentages of populations. Data are expressed as the mean ± SEM using the "nonbiased" standard deviation method. Percent depletion was calculated by normalizing all data to mock-treated controls before calculating means and standard deviations. Significance was defined only according to Student’s t results of untransformed data. Level of significance (P value) is indicated where data are presented.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transgene construction and detection of expression in vivo
The Mafia transgene was assembled by inserting the FKBP–Fas suicide construct [19 ] into a modified version of the pGL-2 plasmid, which contained the c-fms promoter with intron 2 and EGFP as described by Sasmono et al. [12 ] (Fig. 1A) . The final construct was verified for proper ligation of cassettes by sequence analysis of overlapping regions in forward and reverse directions at the following junctions: EGFP to IRES, IRES to {Delta}LNGFR, and Fas to pGL-2. Sequence alignment from the Mafia transgene matched published sequences of each cassette. The 11.1-kb transgene was excised from the pGL-2 plasmid and microinjected into the pronuclei of fertilized C57Bl/6J eggs. Pups were screened for the presence of the Mafia transgene in genomic DNA by real-time PCR and for EGFP expression by flow cytometric analysis of peritoneal macrophages. Flow cytometric analysis of EGFP expression in freshly isolated peritoneal cells demonstrated that 84% of the cells from Mafia mice were EGFP+ (Fig. 1B , lower right panel). When EGFP-positive peritoneal macrophages from Mafia mice were also stained with anti-nerve growth factor receptor antibody to identify expression of the {Delta}LNGFR epitope tag of the suicide protein, 78% of freshly isolated peritoneal cells were double-positive for EGFP and {Delta}LNGFR, indicating that proteins for each of the bicistronic mRNAs were constitutively expressed in Mafia peritoneal macrophages (Fig. 1B , upper right panel).

A small population of peritoneal cells from the Mafia mouse exhibited an intermediate level of EGFP expression as observed in Figure 1B , right panels. Within this minor population of EGFPlow-expressing cells, 34.4% were also positive for {Delta}LNGFR, suggesting low-level expression of the Mafia transgene by a subset of the EGFPlow cells. In contrast, 95.1% of the EGFPhigh population coexpressed {Delta}LNGFR (Fig. 1B , upper right panel).

Tissue distribution and cell specificity of Mafia transgene expression
EGFP expression was analyzed in single-cell suspensions from the peritoneum, blood, spleen, lung, lymph node, thymus, bone marrow, and brain to determine the distribution of EGFP+ cells in various tissues from Mafia mice (Fig. 2 ). All tissues displayed a population of EGFP+ cells consistent with the expected percentage of macrophages in those tissues. The brightness of EGFP expression varied among different tissues, and the brightest cells were seen in the peritoneum and lung, where cells were five to 10 times brighter than for other tissues analyzed.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Distribution of EGFP expression. (A–H) Cells from tissues of a Mafia mouse (shaded) and a WT mouse (solid line) were analyzed by flow cytometry for EGFP expression. Histograms were graphed from events gated by forward- and side-scatter (FSC and SSC, respectively) to the region where macrophages and lymphocytes would be found.

 
Single-cell suspensions from selected tissues of Mafia mice or C57Bl/6J (WT) mice were stained with antibodies specific for macrophages, DC, neutrophils, T cells, or B cells to determine if EGFP expression was restricted to the macrophage population (Fig. 3 ). Between 70 and 76% of F4/80+ macrophages in the peritoneal cavity and the lungs of Mafia mice expressed EGFP compared with less than 3% nonspecific background fluorescence of WT F4/80+ cells in the EGFP region. Among the CD11b+ cells, 97.1% of peritoneal cells and 76.3% of lung cells coexpressed EGFP in Mafia mice. GR1+ cells in the peritoneal cavity did not express EGFP; however, 56% of GR1+ cells in a whole-lung cell suspension expressed EGFP in Mafia mice at a lower fluorescence level than the EGFP+ F4/80+ population. GR1+ cells in the lung were also CD11b+ and F4/80+. Splenic CD11c+ cells in Mafia mice expressed EGFP levels higher than that of WT CD11c+ cells but at a lower fluorescence than F4/80+ EGFP+ cells in a peritoneal lavage. CD11c+ cells in the lung also expressed EGFP and were negative for CD11b and GR1. EGFP expression was not detected in splenic B220+ or CD3+ populations.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 3. Specificity of EGFP expression. Cells from a Mafia mouse (shaded) or from a WT mouse (solid line) were stained for specific cell-surface markers. Histograms of EGFP expression were graphed from events gated by cells in the regions expressing (A) F4/80, (B) CD11b, (C) GR-1 from a peritoneal lavage, (D) CD11c, (E) B220, and (F) CD3{varepsilon} from spleen and (G) F4/80, (H) CD11b, (I) GR1, and (J) CD11c from whole lung.

 
Induction of macrophage depletion in Mafia mice
The ability of the Mafia transgene to allow for induction of macrophage depletion via the FKBP–Fas suicide gene was initially analyzed in vitro. Peritoneal cells from Mafia or WT mice were cultured in the presence or absence of 10 nM AP20187 to determine if the dimerizer could activate the FKBP–Fas suicide gene and deplete macrophages in vitro. EGFP+ peritoneal cells from Mafia mice were decreased by 81.8% compared with untreated Mafia cells after 48 h culture in the presence of the dimerizer (not shown). Flow cytometric analysis of the peritoneal macrophage population, as indicated by FSC and SSC parameters, showed a parallel decrease of macrophage-like cells by 78.3% in cultures treated with dimerizer. In contrast, AP20187 treatment of WT peritoneal cells had no effect on the viability of macrophages in vitro. Dose-response studies on sorted EGFP+ peritoneal cells from Mafia mice demonstrated an IC50 of the AP20187 dimerizer of 0.37 nM, which is within the range of sensitivity of other cell types transfected with this suicide gene [19 ].

In preliminary in vivo studies, it was observed that single injections with doses as high as 50 mg/kg were insufficient to cause major depletion of macrophages in Mafia mice within 72 h of treatment. However, with administration of daily injections with a dose of 10 mg/kg, depletion became evident as early as 24 h after the third injection. To determine the ability of the dimerizer to deplete macrophages in vivo, Mafia mice were given intraperitoneal (i.p.) injections for 5 consecutive days with AP20187 at a dose of 10 mg/kg or with a diluent control. It should be noted that AP20187-treated Mafia mice lost an average of 18.1 ± 6.7% of their body weight by the end of the 5-day treatment protocol. However, mock-treated Mafia, AP20187-treated WT, and mock-treated WT mice did not lose weight during the study period, indicating that the dimerizer was not generally toxic and that weight loss in dimerizer-treated Mafia mice was likely related to macrophage depletion in vivo.

Twenty-four hours after the last of the five injections, the percentage of EGFP+ cells was analyzed by flow cytometry in single-cell suspensions from a variety of tissues (Table 1 ). Viable F4/80+ cells in the peritoneal lavage decreased 86.9 ± 7.5% after the 5-day treatment. This change is different (P<0.05) from the percent loss when determined only by EGFP detection as presented in Table 1 . Compared with diluent control, AP20187 treatment reduced EGFP+ cells by greater than 90% in bone marrow and peritoneum and greater than 70% in the blood, spleen, lung, and thymus. No significant loss of EGFP+ cells was observed in the brain or lymph nodes by flow cytometry. At 24 h post-treatment, spleens exhibited no significant changes in B220+ or CD3+ cell percentages within the CD45+ population.


View this table:
[in this window]
[in a new window]
  		Table 1. Percent EGFP+ Cells in Mafia Mice Following Treatment with AP20187 Dimerizer

 
In lymph nodes, B220+ cells increased from 19 ± 6.3 to 39.6 ± 8.5% of the CD45+ population (significant at P<0.01), and CD3+ cells decreased from 66.0 ± 7.0 to 43.5 ± 13.5% (significant at P<0.05). The thymic CD4/CD8 double-positive population decreased significantly (P<0.01) from 65.3 ± 2.0 to 10.3 ± 1.5%, and single-positive CD4 cells increased from 10.0 ± 1.9 to 48.0 ± 10.7% (P<0.01). There was no significant change in single-positive CD8 cells.

To determine if depleted EGFP+ cells would be re-established in tissues after completion of the dimerizer treatment protocol, Mafia mice were rested for 7 days after the last dimerizer injection and then analyzed by flow cytometry for expression of EGFP (Table 1) . Although the level of depletion was still significant on day 7, the percentage of EGFP+ cells was beginning to return in some tissues. The greatest return of EGFP+ cells occurred in the bone marrow and peritoneal cells. In contrast to the depletion of macrophages, CD11c+ splenic DC exhibited a delay in depletion. Flow cytometric analysis CD11c+ splenic cell populations at 24 h and 7 days after the 5-day AP20187 exposure indicated that DC depletion increased from 48.3 ± 13.2% to 96.8 ± 2.2% of CD11c+ cells, whereas macrophage depletion in the spleen showed no increase during that time.

As an EGFPlow population of cells was observed in the peritoneum of Mafia mice, the effect of dimerizer treatment on this population was also evaluated. At 24 h post-treatment, the low-expressing EGFP+ population in the peritoneum increased to 38.0 ± 16.3% in AP20187-treated Mafia mice, which was significantly higher (P<0.001) than the 11.8 ± 5.3% in mock-treated mice. The EGFPlow cells in the peritoneum remained elevated at 1 week post-treatment with no significant change from the increase observed at 24 h post-treatment. Fluorescent antibody staining of the EGFPlow cells in AP20187-treated mice revealed that 94.4 ± 1.9% of these cells were GR1+. The population of GR1+ GFP– cells observed in untreated mice (Fig. 3) was still present in AP20187-treated mice, and 3.1 ± 5.2% of the GFPhigh cells were GR1+. To better understand the nature of the EGFPlow cells, the EGFPhigh and EGFPlow cells from untreated and AP20187-treated Mafia mice were sorted, and cytospin preparations were observed for morphologic characterisitics (Fig. 4 ). The EGFPhigh and EGFPlow subsets from normal Mafia mice had characteristics of normal macrophage morphology. The EGFPhigh cells from dimerizer-treated mice exhibited a mononuclear morphology; however, most cells displayed highly vesiculated cytoplasms with membrane blebbing characteristic of cells undergoing apoptosis. Conversely, the EGFPlow cells from treated Mafia mice exhibited characteristic neutrophil morphology including band neutrophils. By flow analysis, only 3.4 ± 1.2% of the EGFPlow neutrophils in treated Mafia mice expressed {Delta}LNGFR.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 4. Cytospin preparations of sorted EGFP+ cells. Cells were obtained from untreated Mafia-sorted populations of (A) EGFPhigh and (B) EGFPlow cells and from AP20187-treated Mafia-sorted populations of (C) EGFPhigh and EGFPlow cells. Cytospin samples were stained with DiffQuick.

 
IHC with anti-GFP antibody was used to visualize macrophage depletion in Mafia mice (Fig. 5 ). Macrophages were clearly evident in the lung, spleen, and liver of all diluent-treated Mafia mice with similar densities to WT mice. In contrast, macrophage populations in Mafia mice were dramatically reduced following the dimerizer treatment protocol. IHC in the lung indicated that alveolar and interstitial macrophages were depleted and that some residual EGFP+ cells appeared to be present within the interstitium of the lung. The greatest concentration of EGFP+ cells in the spleens of mock-treated mice was in the red pulp areas and marginal zones of the follicles. AP20187 treatment greatly reduced the number of EGFP+ cells in all areas of the spleen with most of the residual EGFP+ cells observed in the red pulp. EGFP+ cells displaying morphologic features of Kupffer cells in liver sinusoids were present in all diluent-treated Mafia mice, whereas only rare EGFP+ cells were seen in liver sections from dimerizer-treated mice.



View larger version (136K):
[in this window]
[in a new window]
  		Figure 5. IHC detection of EGFP expression and depletion within tissues. Mafia miced were treated with diluent or 10 mg/kg AP20187 for 5 days, and tissue sections from (A, B) spleen, (C, D) liver, and (E, F) lung were stained with anti-GFP–biotin and antibiotin–horseradish peroxidase. Macrophages stained dark brown (arrows).

 
Epidermal sheets were collected from the ears of WT mice and from mock-treated and 5-day AP20187-treated Mafia mice rested 1 week. Wet mounts were observed by epifluorescence (Fig. 6 ). Fluorescent cells were not detected in epidermal sheets from WT mice. Langerhans cells were clearly visible as EGFP+ cells in mock-treated Mafia mice. In contrast, EGFP+ cells with normal morphological appearance were not evident in AP20187-treated Mafia mice. Diffuse, weakly EGFP+ cell bodies, which lacked DC morphology, were sometimes visible in epidermal sheets from treated mice and appeared to be dead or dying Langerhans cells.



View larger version (123K):
[in this window]
[in a new window]
  		Figure 6. Epifluoresence of epidermal sheet wet-mount preparations from ears. Samples were collected from mock-treated WT mice (A, B), mock-treated Mafia mice (C, D), and AP20187-treated Mafia mice (E, F) 1 week post-treatment. (F) Arrows identify residual cell bodies that expressed low levels of EGFP.

 
Bacterial infection in macrophage-depleted Mafia mice
Although significant levels of macrophage depletion occurred after dimerizer treatment, it was important to confirm that the level of depletion achieved could disrupt the biological function of the macrophage-phagocyte system. The ability of bacteria to be cleared from the blood and to grow in organ tissues was compared in Mafia and WT mice after dimerizer treatment (Fig. 7 ).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 7. Macrophage depletion and bacterial infection. (A) Bacterial clearance from blood. AP20187-treated and mock-treated WT and Mafia mice were challenged with Y. pestis by i.v. injection. Mice were killed after 15 min, and blood samples were plated to determine the viable bacteria numbers as CFU per ml blood. CFU of AP20187-treated Mafia mice were significantly higher than the three control groups, whether compared individually or collectively (P=0.018). (B) Bacterial growth in liver. At various time-points after i.v. infection of Y. pestis, homogenized liver extracts from AP20187-treated and mock-treated Mafia mice were plated to determine the viable numbers as CFU per gram of liver. *, P< 0.05; #, P< 0.01. N.S., Not significant.

 
Twenty-four hours after the last injection with dimerizer, mice were injected i.v. with the conditionally virulent Y. pestis strain KIM5. Blood samples were plated for viable bacteria at 15 min after infection (Fig. 7A) . WT mice effectively cleared the bacteria, regardless of whether they were pretreated with AP20187 or diluent. Mafia mice mock-treated with diluent cleared the inoculated bacteria as efficiently as WT mice. In contrast, significantly higher levels of Y. pestis (P<0.05) were detected in the blood of dimerizer-treated Mafia mice.

In a separate study, the bacterial load was determined in livers of AP20187-treated and mock-treated Mafia mice at various times after infection (Fig. 7B) . Fifteen minutes after infection, the amount of bacteria in the liver was similar for mock-treated and macrophage-depleted mice. However, at all subsequent time-points, the amount of bacteria in the liver was significantly higher in AP20187-treated Mafia mice.

Abnormalities in Mafia mice after macrophage/DC depletion
Treatment of Mafia mice with AP20187 caused the development of several gross abnormalities, which may suggest unexpected roles for macrophages and/or DC during homeostatic conditions in the immune system. Mafia mice experienced severe splenomegaly, lymphadenopathy, and thymic atrophy following depletion of macrophages and DC. Twenty-four hours after the last injection of AP20187, spleens were significantly enlarged 1.6 ± 0.4-fold compared with Mafia mice treated with diluent. Splenomegaly continued following completion of the dimerizer treatment protocol, increasing to 7.6 ± 2.3-fold larger spleens 7 days after treatment. Histological evaluation indicated that extensive extramedullary hematopoiesis was occurring in the spleens of macrophage/DC-depleted mice (Fig. 8A ). At 24 h and 7 days after the depletion protocol, 100% of spleens exhibited islands of extramedullary hematopoiesis with megakaryocytosis and severe or moderate disruption of follicular morphology. Islands of extramedullary hematopoiesis were also observed in the livers of depleted Mafia mice; however, the extent of tissue morphology disruption was not as severe as in the spleen. Spleens from diluent-treated Mafia mice displayed only occasional and very minor extramedullary hematopoiesis, which was no greater than that observed in diluent-treated or AP20187-treated WT mice, indicating that the hematopoietic response in AP20187-treated Mafia mice was related to the systemic depletion of macrophages and DC.



View larger version (127K):
[in this window]
[in a new window]
  		Figure 8. Altered splenic structure and peritoneal adhesions. (A) Spleens from mock-treated and 10 mg/kg AP20187-treated Mafia mice were harvested 24 h after the 5-day treatment and stained with hematoxalin and eosin to observe splenic structure. Disruption of follicles was observed in the treated mouse (brackets), and zones of extramedullary hematopoiesis were also identifiable (arrows). (B) Peritoneal adhesions firmly attached the liver of a treated Mafia mouse to its diaphragm (left panel) and the spleen to the peritoneal wall (right panel). The adhesions developed 1–2 weeks post-treatment and were opaque and fibrous in appearance (arrows).

 
Bone marrow samples were analyzed for lineage-negative (lin–; CD19–, GR1–, CD3–, TER119–, CD5–), c-kit+ sca-I+ cells to determine if hematopoietic stem cells were affected by AP20187 treatment. Within this population, 13.9 ± 2.8% of the cells expressed EGFP. The lin– c-kit+ sca-I+ population was reduced by 18.5 ± 16.4% 1 day post-depletion, but this difference was not significantly different from controls. One week post-depletion, the lin– c-kit+ sca-I+ population decreased by 35.8 ± 17.8% (P<0.05); however, stem-cell depletion never approached the level of depletion seen for macrophages and DC. In healthy Mafia mice, 17.9 ± 6.5% of the TER119+ erythrocyte precursor population in the bone marrow expressed EGFP. A reduction of 84.5 ± 9.1% in TER119+ bone marrow cells was observed in AP20187-treated Mafia mice 1 day after the last injection of dimerizer, and a 97.2 ± 1.3% decrease was evident 1 week post-depletion compared with mock controls. The loss of TER119+ cells from the bone marrow affected the erythropoietic capacity in Mafia mice, as at the end of the depletion protocol, hematocrits were reduced by 11.1 ± 4.1% (P<0.05) in AP20187-treated Mafia mice compared with mock-treated controls.

Enlarged lymph nodes were also evident in macrophage/DC-depleted mice. In histological sections, the lymphadenopathy appeared as a result of an increase in cell number rather than fluid accumulation. However, the morphology of follicular, paracortical, and medullary regions of the lymph nodes appeared normal, and there was no evidence of extracellular hematopoiesis.

Severe thymic atrophy was observed in AP20187-treated Mafia mice but not in the diluent-treated Mafia or any group of treated WT mice. Twenty-four hours after the depletion protocol, thymi were decreased in weight 60.3 ± 18.7% compared with those from diluent-treated Mafia mice. Moreover, 7 days after depletion, fewer than 25% of AP20187-treated Mafia mice possessed any evidence of an identifiable thymus.

An unexpected abnormality was the development of peritoneal adhesions in ~80% of adult Mafia mice within 7 days after completion of the depletion protocol. The most prominent site of peritoneal adhesions was between the superior surface of the liver and the inferior surface of diaphragm (Fig. 8B) . Evidence of adhesions was observed at many other locations within the peritoneum, including between the lateral or dorsal side of the spleen to the peritoneal wall. In spite of the frequency of observation for peritoneal adhesions, pericardial adhesions were rarely observed, although the pericardial sac appeared increased in thickness and more opaque following AP20187 treatment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Studies presented here describe a new, transgenic approach that enables systemic depletion of macrophages in adult C57Bl/6J mice. The Mafia suicide gene consists of the {Delta}LNGFR–FKBP–Fas construct expressed under control of the c-fms promoter containing intron 2 for specific expression in cells of the macrophage lineage. Maximal depletion of macrophages in vivo required multiple daily injections of the AP20187 dimerizer. Depletion appeared to be reversible in that EGFP+ cells began to return by 7 days after cessation of treatment. Expression of the Mafia transgene occurred primarily in macrophages and DC. A similar pattern of expression was previously observed in transgenic mice containing the c-fms promoter and EGFP [12 ]. Mafia mice did not express EGFP in B cells or T cells as determined by flow cytometry analysis of B220 and CD3 expression.

Cytospin preparations of sorted EGFPlow cells in our laboratory indicated that neutrophils are the primary cell type present in the EGFPlow population of treated Mafia mice. The large influx of these granulocytes was not surprising, as macrophages have been shown to release neutrophil chemotactic factors when undergoing Fas-induced apoptosis [22 ]. In addition, it was not unexpected to see some EGFP expression in neutrophils, as macrophages and granulocytes share a common progenitor cell, which is dependent on CSF-1 [5 , 6 , 12 ]. However, mature granulocytes do not express the CSF-1 receptor [23 ], suggesting that EGFP protein in neutrophils was not likely a result of continued transcription of the transgene. Sasmono et al. [12 ] also detected c-fms-driven EGFP expression in tissue-resident granulocytes, although previous studies have shown no expression of c-fms mRNA or protein in these cells [24 , 25 ]. These authors suggested that the half-life of EGFP is long and that EGFP protein may consequently persist in mature granulocytes, although the c-fms promoter is inactive. Three separate populations of neutrophils were observed in Mafia mice. In untreated Mafia mice, the only population observed was GR1+ EGFP–. However, following treatment, two EGFPlow populations of neutrophils were also observed. The predominant EGFPlow fraction did not coexpress the suicide gene, as indicated by the lack of the {Delta}LNGFR epitope tag and is presumed to be derived from an earlier influx of neutrophils from the bone marrow in which the suicide protein has decayed. A minor population of EGFPlow neutrophils (less than 3% of the EGFPlow population) also expressed the suicide protein and is assumed to be the most recent emigrants from the marrow in which the suicide protein has yet to turn over. These data suggest that the membrane-bound suicide protein, as with the CSF-1 receptor, may have a higher turnover rate than the cytoplasmic EGFP protein. It is important to note that dimerizer treatment only depleted macrophages and DC, and the level of granulocytes in the peritoneal cavity increased.

The c-fms gene is expressed in macrophages and DC [20 ]; thus, expression of EGFP and the FKBP–Fas suicide gene in DC of Mafia mice was expected. Likewise, in studies by Sasmono et al. [12 ], transgenic mice expressing EGFP under the c-fms promoter expressed EGFP in DC in the spleen and skin. CD11c+ DC in the spleen were observed to express the Mafia transgene; however, the level of EGFP expression was tenfold lower than in macrophages. Langerhans cells in epidermal sheets from Mafia mice expressed visible amounts of EGFP, and peritoneal treatment with AP20187 caused nearly complete loss of Langerhans cells by 1 week post-treatment. It is possible that dimerizer treatment induced migration of Langerhans cells from the skin; however, we suggest that migration is not the cause of the loss of these cells, as a concomitant increase in DC within lymph nodes and spleen was not observed and as areas of the epidermal sheets contained what appeared to be amorphous residual bodies, which were weakly EGFP+ and were likely to be dead or dying Langerhans cells. Hume et al. [23 ] also demonstrated c-fms EGFP transgene expression in Langerhans cells in skin, presumably immature DC, and in myeloid DC in lymph nodes.

Activation of immature DC can down-regulate expression of the c-fms gene, which may help in the commitment of immature DC into functional APC [24 , 26 ]. It is possible that the CD11c+ EGFP+ cells analyzed in the spleen of Mafia mice are of a more mature or activated phenotype, which has down-regulated c-fms expression, causing a lower level of EGFP expression than observed in macrophages. As extensive analysis of DC subpopulations has not yet been performed in Mafia mice, it is unknown whether EGFP expression is low in all DC populations. Reduced expression of the Mafia transgene did not prevent elimination of CD11c+ DC by AP20187 treatment; however, the elimination of DC in the spleen was slower than the loss of macrophages. It is possible that the lower transgene expression level in DC is responsible for the slower rate of cell depletion. Kamath et al. [27 ] demonstrated that CD8+ DC in the spleen have a 3-day turnover rate, which makes it equally possible that depletion of macrophages removed a source of DC precursors, and the natural turnover of DC leads to an effective loss. Regardless of the mechanism, the depletion level of DC in the spleen and Langerhans cells in the skin was dramatic, approaching 100% by day 7 after completion of dimerizer treatment.

EGFP+ cells were reduced in all organs evaluated, except for the brain and lymph nodes. Currently, no information has been reported regarding the in vivo distribution of AP20187. It is possible that the dimerizer cannot effectively penetrate the blood-brain barrier and that another mode of administration may be necessary for effective distribution to the brain. Similarly, AP20187 may not reach the lymphatic drainage in sufficient amounts to induce depletion of EGFP+ cells in lymph nodes. Preliminary studies of alternate routes of AP20187 treatment suggest that at least partial depletion (~50%) can be achieved in the brain using an i.v. route. Additional studies will be needed to optimize the dimerizer treatment protocol to achieve depletion in lymph nodes.

The cause of thymic atrophy in AP20187-treated Mafia mice is not yet determined. Flow cytometric analysis showed that the predominant cell population lost at 24 h post-treatment was the CD4+CD8+ double-positive thymocyte fraction, suggesting that thymic atrophy in macrophage/DC-depleted mice may be a result of a stress response during depletion. Glucocorticoid-dependent and -independent processes can cause thymic atrophy [28 , 29 ]. Lipopolysaccharide can induce apoptosis of lymphocytes in a tumor necrosis factor {alpha} (TNF-{alpha})-dependent manner [30 ], and liver injury by Fas-induced apoptosis has been shown to result in elevated TNF-{alpha} serum levels [31 ]. However, analysis of serum TNF-{alpha} by enzyme-linked immunsorbent assay indicated that TNF-{alpha} levels were not significantly different in AP20187-treated Mafia mice compared with other treatment groups (data not shown), suggesting that thymic atrophy was not caused by a release of TNF-{alpha}. Further investigation is needed to determine if glucocorticoids are released during dimerizer treatment to contribute to thymic atrophy in Mafia mice or whether atrophy occurs as a direct result of macrophage/DC depletion.

Billia et al. [32 ] reported expression of c-fms in TER119+ erythroid precursor cells as well as in multipotent stem cells, which is in agreement with the EGFP expression in TER119+ cells and lin– c-kit+ sca-I+ cells in the bone marrow of Mafia mice. Considering that only 14% of lin– c-kit+ sca-I+ cells expressed EGFP by flow cytometric analysis, the significant loss in this population at 1 week post-depletion may be a result of more than the depletion of EGFP+ cells by dimerizer. Likewise, although only 18% of TER119+ bone marrow cells expressed EGFP, 97% of all TER119+ erythrocyte precursors were lost by 1 week post-depletion. These observations may indicate that resident macrophages and/or DC could act as important regulators of hematopoiesis and that in their absence, hematopoietic cell development in the marrow may not proceed normally. Studies are in progress to address the role of resident marrow macrophages and DC in the regulation of hematopoeisis.

Depletion successfully impaired the ability of Mafia mice to clear bacteria from the blood and to control bacterial growth in the liver. It has previously been shown that mice with competent immune systems clear bacteria such as Listeria monocytogenes, Salmonella typhimurium, and Yersinia enterocolitica from blood within 15 min of i.v. exposure [33 , 34 ]. Bacterial clearance from blood is assumed to be primarily a result of macrophages [33 ], and control of bacterial growth in tissues is dependent in part on resident and inflammatory macrophages. Dimerizer-treated Mafia mice did not clear the challenge bacteria from the blood at the same rate as mock-treated mice. Although bacterial clearance from the blood was altered, bacteria still accumulated in the liver, and AP20187-treated mice exhibited a more aggressive bacterial growth pattern in the liver compared with mock-treated Mafia, thus demonstrating a functional loss in vivo as a result of macrophage depletion. Additional functional studies are currently underway to investigate other aspects of the inductive and effector phases of the immune response in macrophage-depleted Mafia mice.

Previous reports of knockouts of the CSF-1 gene (Csf1op/Csf1op) or the CSF-1 receptor gene (Csf1r–/Csf1r–) have demonstrated embryonic deficiencies in macrophage development [11 , 35 ]. In both studies, a variety of gross abnormalities were evident in the off-spring mice, including low body weight, low growth rate, skeletal abnormalities, poor fertility, lack of tooth development, and developmental problems in neural and dermal tissues. None of these phenotypes was present in Mafia mice, and mice remain normal throughout adulthood, as macrophages remain at normal levels until administration of dimerizer. However, following dimerizer treatment, several abnormalities were evident in Mafia mice, which are likely related to the loss of macrophages and DC.

Preliminary studies not presented in this report have indicated that depleted Mafia mice display reduced bone resorptive activity within 7 days following dimerizer treatment. A similar loss in activity was seen in Csf1op/Csf1op) and (Csf1r–/Csf1r–) mice, which led to increased bone density in the first few months of age. Studies in Mafia mice exposed to prolonged dimerizer treatment will be necessary to determine the effects of chronic depletion on bone metabolism.

An intriguing observation in (Csf1op/Csf1op) and (Csf1r–/Csf1r–) mice is the finding of CSF-1-independent macrophage populations. Although circulating levels of CSF-1 were undetectable in (Csf1op/Csf1op) mice, some tissues still exhibited a small number of macrophages, which were interpreted as the existence of a macrophage population whose development is independent of CSF-1. Not all F4/80+ macrophages in Mafia mice were EGFP+. Furthermore, macrophage depletion was not complete in all tissues from dimerizer-treated Mafia mice, and a comparison of F4/80+ versus EGFP+ populations demonstrated that a greater portion of F4/80+ cells survived the treatment than EGFP+ cells. Whether the failure to completely eliminate macrophages in some tissues is a result of a subpopulation of macrophages in Mafia mice, which never expressed or lost the ability to express c-fms and/or the suicide gene, remains to be determined. It is interesting that DC-rich regions in lymphoid organs and in skin were unaltered in (Csf1op/Csf1op) mice, suggesting that c-fms expression may not be necessary for maintenance of DC.

An unexpected finding was the development of peritoneal adhesions in most Mafia mice following dimerizer treatment. Peritoneal adhesions are an important complication associated with abdominal or pelvic surgery, occurring in more than 90% of patients after major abdominal surgery and in 55–100% of the patients after pelvic surgery [36 ]. The role of macrophages in the pathogenesis of adhesions is unclear, and studies with conflicting conclusions have reported that macrophages protect from or promote adhesion development [37 , 38 ]. Induction of adhesions is thought to occur at sites of injury to the mesothelial layer covering peritoneal tissues. Deposition of fibrin at injured sites and a concurrent decrease in fibrinolytic activity contribute to adhesion development. Macrophages appear to be a major source of fibrinolytic activity in the peritoneal cavity, and this activity is decreased by peritoneal trauma [39 , 40 ]. In one study, post-operative adhesion formation was reduced in a rabbit model when peritoneal macrophage numbers were increased before peritoneal injury [41 ]. The mesothelium appears to play an important role in the regulation of coagulation and fibrin formation on serosal surfaces [42 , 43 ]. Resident peritoneal macrophages constitutively produce high levels of plasminogen-activator inhibitor [44 ] and thus, under homeostatic conditions, would be protective against fibrin deposition in the peritoneal cavity. The observation of adhesion development in dimerizer-treated Mafia mice suggests that macrophages play a protective role and that depletion contributes to the development of peritoneal adhesions. Mock-treated Mafia mice as well as AP20187-treated WT mice did not develop adhesions, and the adhesions in treated Mafia mice occurred in regions not directly at the site of i.p. injection. Adhesion formation has not been reported in mice treated with chlodronate for macrophage depletion. It is possible that i.p.injection of AP20187 might more efficiently eliminate peritoneal macrophages and macrophages residing within the mesothelium, allowing a threshold to be reached that permits adhesion development. Preliminary histological evaluation of liver and spleen in dimerizer-treated Mafia mice indicated that the mesothelial layer was disrupted in the areas of adhesion development but appeared intact at other regions of the mesothelium. It is unknown whether this disruption was an initiation event for adhesion formation or whether it was a consequence of adhesion development. However, it remains a possibility that dimerizer treatment also causes injury to mesothelial cells and that adhesions develop as a result of a combined effect of macrophage loss and mesothelial damage. Additional studies will be required to determine if macrophages resident in the peritoneal cavity and/or mesothelial lining are playing a direct role in protecting the peritoneum from adhesion formation during homeostatic conditions and whether mesothelial cells are being injured during dimerizer treatment.

In conclusion, systemic depletion of macrophages and DC can be transiently induced in Mafia mice following cell-specific activation of transgene-associated Fas activity. Studies with this new, transgenic mouse line should enable elucidation of macrophage/DC functions in tissues not easily affected by other depletion approaches. The constitutive expression of EGFP in these cells also allows easy identification in tissues, as well as flow cytometric sorting to purify macrophages and DC from any tissue. Adoptive transfer of purifed EGFP+ cells, monitoring of cell migration, and subsequent depletion of transferred cells should enhance the ability of investigators to better define the immunological and physiological functions of macrophages and DC in vivo.


   ACKNOWLEDGEMENTS
 
We thank Dr. David Hume (Univ. of Queensland, Australia) for the c-fms promoter/EGFP plasmid and Dr. Tim Clackson (Ariad Pharmaceuticals, Inc., Cambridge, MA) for the FKBP-Fas construct. We are also grateful to Ariad Pharmaceuticals, Inc. for supplying the AP20187 dimerizer. This work was supported by the following NIH grants: HL69459 and HL57399. SHB and EJK were supported by the NIH training grant: 5T32AI049795.

Received September 24, 2003; revised December 16, 2003; accepted December 18, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schindler, L., Brucher, J., Kirchner, H. (1984) Protection of mice against infection with mouse hepatitis virus type 3 by injection of silica Immunobiology 166,62-71[Medline]
  2. Ishizaka, S., Kuriyama, S., Tsujii, T. (1989) In vivo depletion of macrophages by desulfated iota-carrageenan in mice J. Immunol. Methods 124,17-24[CrossRef][Medline]
  3. Mizgerd, J. P., Molina, R. M., Stearns, R. C., Brain, J. D., Warner, A. E. (1996) Gadolinium induces macrophage apoptosis J. Leukoc. Biol. 59,189-195[Abstract]
  4. Buiting, A. M., Van Rooijen, N. (1994) Liposome mediated depletion of macrophages: an approach for fundamental studies J. Drug Target. 2,357-362[Medline]
  5. Scott, E. W., Simon, M. C., Anastasi, J., Singh, H. (1994) Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages Science 265,1573-1577[Abstract/Free Full Text]
  6. McKercher, S. R., Torbett, B. E., Anderson, K. L., Henkel, G. W., Vestal, D. J., Baribault, H., Klemsz, M., Feeney, A. J., Wu, G. E., Paige, C. J., Maki, R. A. (1996) Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities EMBO J. 15,5647-5658[Medline]
  7. Sauer, B., Henderson, N. (1990) Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase New Biol 2,441-449[Medline]
  8. Holness, C. L., da Silva, R. P., Fawcett, J., Gordon, S., Simmons, D. L. (1993) Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family J. Biol. Chem. 268,9661-9666[Abstract/Free Full Text]
  9. Roberts, W. M., Shapiro, L. H., Ashmun, R. A., Look, A. T. (1992) Transcription of the human colony-stimulating factor-1 receptor gene is regulated by separate tissue-specific promoters Blood 79,586-593[Abstract/Free Full Text]
  10. Yue, X., Favot, P., Dunn, T. L., Cassady, A. I., Hume, D. A. (1993) Expression of mRNA encoding the macrophage colony-stimulating factor receptor (c-fms) is controlled by a constitutive promoter and tissue-specific transcription elongation Mol. Cell. Biol. 13,3191-3201[Abstract/Free Full Text]
  11. Cecchini, M. G., Dominguez, M. G., Mocci, S., Wetterwald, A., Felix, R., Fleisch, H., Chisholm, O., Hofstetter, W., Pollard, J. W., Stanley, E. R. (1994) Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse Development 120,1357-1372[Abstract]
  12. Sasmono, R. T., Oceandy, D., Pollard, J. W., Tong, W., Pavli, P., Wainwright, B. J., Ostrowski, M. C., Himes, S. R., Hume, D. A. (2003) A macrophage colony-stimulating factor receptor (CSF-1R)-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse Blood 101,1155-1163[Abstract/Free Full Text]
  13. Dzierzak, E., Daly, B., Fraser, P., Larsson, L., Muller, A. (1993) Thy-1 tk transgenic mice with a conditional lymphocyte deficiency Int. Immunol. 5,975-984[Abstract/Free Full Text]
  14. Bryson, J. S., Lake-Bullock, H., Pflugh, D. L., Jennings, C. D., Stuart, P. M., Caywood, B. E., Kaplan, A. M. (1995) In vivo reactivity of T cell clones isolated from mice with syngeneic graft-versus-host disease Transplantation 60,171-178[Medline]
  15. Heyman, R. A., Borrelli, E., Lesley, J., Anderson, D., Richman, D. D., Baird, S. M., Hyman, R., Evans, R. M. (1989) Thymidine kinase obliteration: creation of transgenic mice with controlled immune deficiency Proc. Natl. Acad. Sci. USA 86,2698-2702[Abstract/Free Full Text]
  16. Salomon, B., Lores, P., Pioche, C., Racz, P., Jami, J., Klatzmann, D. (1994) Conditional ablation of dendritic cells in transgenic mice J. Immunol. 152,537-548[Abstract]
  17. Ashkenazi, A., Dixit, V. M. (1998) Death receptors: signaling and modulation Science 281,1305-1308[Abstract/Free Full Text]
  18. Spencer, D. M., Belshaw, P. J., Chen, L., Ho, S. N., Randazzo, F., Crabtree, G. R., Schreiber, S. L. (1996) Functional analysis of Fas signaling in vivo using synthetic inducers of dimerization Curr. Biol. 6,839-847[CrossRef][Medline]
  19. Thomis, D. C., Marktel, S., Bonini, C., Traversari, C., Gilman, M., Bordignon, C., Clackson, T. (2001) A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease Blood 97,1249-1257[Abstract/Free Full Text]
  20. Rieser, C., Ramoner, R., Bock, G., Deo, Y. M., Holtl, L., Bartsch, G., Thurnher, M. (1998) Human monocyte-derived dendritic cells produce macrophage colony-stimulating factor: enhancement of c-fms expression by interleukin-10 Eur. J. Immunol. 28,2283-2288[CrossRef][Medline]
  21. Straley, S. C., Harmon, P. A. (1984) Growth in mouse peritoneal macrophages of Yersinia pestis lacking established virulence determinants Infect. Immun. 45,649-654[Abstract/Free Full Text]
  22. Hohlbaum, A. M., Gregory, M. S., Ju, S-T., Marshak-Rothstein, A. (2001) Fas ligand engagement of resident peritoneal macrophages in vivo induces apoptosis and the production of neutrophil chemotactic factors J. Immunol. 167,6217-6224[Abstract/Free Full Text]
  23. Hume, D. A., Ross, I. L., Himes, S. R., Sasmono, R. T., Wells, C. A., Ravasi, T. (2002) The mononuclear phagocyte system revisited J. Leukoc. Biol. 72,621-627[Abstract/Free Full Text]
  24. Sester, D. P., Beasley, S. J., Sweet, M. J., Fowles, L. F., Cronau, S. L., Stacey, K. J., Hume, D. A. (1999) Bacterial/CpG DNA down-modulates colony stimulating factor-1 receptor surface expression on murine bone marrow-derived macrophages with concomitant growth arrest and factor-independent survival J. Immunol. 163,6541-6550[Abstract/Free Full Text]
  25. Fowles, L. F., Stacey, K. J., Marks, D., Hamilton, J. A., Hume, D. A. (2000) Regulation of urokinase plasminogen activator gene transcription in the RAW264 murine macrophage cell line by macrophage colony-stimulating factor (CSF-1) is dependent upon the level of cell-surface receptor Biochem. J. 347,313-320
  26. Chomarat, P., Banchereau, J., Davoust, J., Palucka, A. K. (2000) IL-6 switches the differentiation of monocytes from dendritic cells to macrophages Nat. Immunol. 1,510-514[CrossRef][Medline]
  27. Kamath, A. T., Henrim, S., Battye, F., Tough, D. F., Shortman, K. (2002) Developmental kinetics and lifespan of dendritic cells in mouse lymphoid organs Blood 100,1734-1741[Abstract/Free Full Text]
  28. Jondal, M., Okret, S., McConkey, D. (1993) Killing of immature CD4+ CD8+ thymocytes in vivo by anti-CD3 or 5'-(N-ethyl)-carboxamide adenosine is blocked by glucocorticoid receptor antagonist RU-486 Eur. J. Immunol. 23,1246-1250[Medline]
  29. Krenger, W., Rossi, S., Hollander, G. A. (2000) Apoptosis of thymocytes during acute graft-versus-host disease is independent of glucocorticoids Transplantation 69,2190-2193[CrossRef][Medline]
  30. Norimatsu, M., Ono, T., Aoki, A., Ohishi, K., Tamura, Y. (1995) In-vivo induction of apoptosis in murine lymphocytes by bacterial lipopolysaccharides J. Med. Microbiol. 43,251-257[Abstract]
  31. Tsutsui, H., Adachi, K., Seki, E., Nakanishi, K. (2003) Cytokine-induced inflammatory liver injuries Curr. Mol. Med. 3,545-549[CrossRef][Medline]
  32. Billia, F., Barbara, M., McEwen, J., Trevisan, M., Iscove, N. N. (2001) Resolution of pluripotential intermediates in murine hematopoietic differentiation by global complementary DNA amplification from single cells: confirmation of assignments by expression profiling of cytokine receptor transcripts Blood 97,2257-2268[Abstract/Free Full Text]
  33. Conlan, J. W. (1997) Critical roles of neutrophils in host defense against experimental systemic infections of mice by Listeria monocytogenes, Salmonella typhimurium, and Yersinia enterocolitica Infect. Immun. 65,630-635[Abstract]
  34. Carter, P. B., Collins, F. M. (1974) Experimental Yersinia enterocolitica infection in mice: kinetics of growth Infect. Immun. 9,851-857[Abstract/Free Full Text]
  35. Dai, X. M., Ryan, G. R., Hapel, A. J., Dominguez, M. G., Russell, R. G., Kapp, S., Sylvestre, V., Stanley, E. R. (2002) Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects Blood 99,111-120[Abstract/Free Full Text]
  36. Liakakos, T., Thomako, N., Fine, P. M., Dervenis, C., Young, R. L. (2001) Peritoneal adhesions: etiology, pathophysiology, and clinical significance.Recent advances in prevention and management Dig. Surg. 18,260-273[CrossRef][Medline]
  37. Haney, A. F. (2000) Identification of macrophages at the site of peritoneal injury: evidence supporting a direct role for peritoneal macrophages in healing injured peritoneum Fertil. Steril. 73,988-995[CrossRef][Medline]
  38. DiZerega, G. S., Campeau, J. D. (2001) Peritoneal repair and post-surgical adhesion formation Hum. Reprod. Update 7,547-555[Abstract/Free Full Text]
  39. Fukasawa, M., Campeau, J. D., Girgis, W., Bryant, S. M., Rodgers, K. E., DiZerega, G. S. (1989) Production of protease inhibitors by postsurgical macrophages J. Surg. Res. 46,256-261[Medline]
  40. Loscalzo, J. (1996) The macrophage and fibrinolysis Semin. Thromb. Hemost. 22,503-506[Medline]
  41. Ar’Rajab, A., Dawidson, I., Sentimentes, J., Sikes, P., Harris, R., Mileski, W. (1995) Enhancement of peritoneal macrophages reduces postoperative peritoneal adhesion formation J. Surg. Res. 58,307-312[CrossRef][Medline]
  42. Mutsaers, S. E. (2002) Mesothelial cells: their structure, function and role in serosal repair Respirology 7,171-191[CrossRef][Medline]
  43. Holmdahl, L. (1997) The role of fibrinolysis in adhesion formation Eur. J. Surg. Suppl. 577,24-31
  44. Costelloe, E. O., Stacey, K. J., Antalis, T. M., Hume, D. A. (1999) Regulation of the plasminogen activator inhibitor-2 (PAI-2) gene in murine macrophages.Demonstration of a novel pattern of responsiveness to bacterial endotoxin J. Leukoc. Biol. 66,172-182[Abstract]



This article has been cited by other articles:


Home page
 ScienceHome page
N. C. Peters, J. G. Egen, N. Secundino, A. Debrabant, N. Kimblin, S. Kamhawi, P. Lawyer, M. P. Fay, R. N. Germain, and D. Sacks
In Vivo Imaging Reveals an Essential Role for Neutrophils in Leishmaniasis Transmitted by Sand Flies
Science, August 15, 2008; 321(5891): 970 - 974.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
L. Albee, B. Shi, and H. Perlman
Aspartic protease and caspase 3/7 activation are central for macrophage apoptosis following infection with Escherichia coli
J. Leukoc. Biol., January 1, 2007; 81(1): 229 - 237.
[Abstract] [Full Text] [PDF]