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Department of Immunology, Schering-Plough Research Institute, Kenilworth, NJ 07033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from 8 to 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c+CD11b+CD8a-), lymphoid (CD11c+CD11b-CD8a+), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A major obstacle in these analyses has been the low number of DC in the circulation and in tissues. Most of the studies to date have relied on DC expanded in vitro by treating BM-derived cells ex vivo with various growth factors and cytokines, such as GM-CSF, TNF-
, or IL-4 (2, 3, 4, 5). However, the number of DC subsets expanded under these conditions is restricted to one or a few subsets. A second approach for expansion of DC involves the use of human FL (hFL). FL is a hemopoietic growth factor that promotes the differentiation and mobilization of hemopoietic progenitors and stem cells in vitro (6, 7) and in vivo (8, 9). Treatment of mice with hFL has been shown to lead to the expansion of DC subsets in multiple tissues and peripheral blood (PB) (5, 10, 11, 12). However, this method involves the daily administration of a foreign protein and manipulation of mice.
To overcome these limitations and to provide a model for studying DC biology in vivo, we developed a noninvasive method for expansion of DC subsets. This method consists of conditional expression of the physiological ligand, murine FL (mFL), in multiple tissues of transgenic (Tg) mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate a bidirectional responder transgene, the cDNA encoding mFL (13) was cloned into pBI-G (Clontech, Palo Alto, CA). The mFL cDNA used in this experiment encodes a functionally active type I transmembrane protein that can be processed to a soluble ligand (14, 15, 16). To generate the activator transgene, cDNA for rtTA was subcloned from pTet-On plasmid (Clontech) into the EcoRI sites of an expression vector containing the hCMV enhancer/chicken 
-actin promoter and the rabbit -globin polyadenylation signal (17). Separation of the transgene from vector sequence was accomplished by zonal sucrose gradient centrifugation as previously described (18). Fractions containing the transgene were pooled, microcentrifuged through Microcon-100 filters (Amicon, Beverly, MA), and washed five times with microinjection buffer (5 mM Tris-HCl (pH 7.4), 5 mM NaCl, and 0.1 mM EDTA).
Mice
Ggeneration of mice that carry the activator transgene (19) or the responder transgene was performed using standard techniques as previously described (20). Identification of Tg founders was conducted by PCR analysis using tail DNA, as previously described (21). The activator and responder mice were crossed to generate mice carrying both transgenes. Identification of double-Tg mice was accomplished by amplification of a segment of the rtTA transgene using the following primers: (5'-CGGGTCTACCATCGAGGGCCTGCT-3') and (5'-CCCGGGGAATCCCCGTCCCCCAAC-3') and amplification of a segment of the -galactosidase (-gal) gene (5'-ACCAGCGAATACCTGTTCCGTCATAGC-3' and (5'-AGTAAGGCGGTCGGGATAGTTTTCTTGC-3'). Primers for the endogenous ZP3 gene (5'-CAGCTCTACATCACCTGCCA-3' and 5'-CACTGGGAAGAGACACTCAG-3') were used as an internal control for the amplification reaction. These primers amplify a 242-bp segment of the rtTA transgene, a 791-bp segment of the -gal gene, and a 511-bp segment of the ZP3 gene. PCR conditions were 95°C for 30 s, 60°C for 30 s, and 72°C for 60 s for 30 cycles. The resulting Tg mice were kept under specific pathogen-free conditions. All experiments with mice followed institutional guidelines.
Bone marrow (BM) transfers
BM from green fluorescent protein (GFP) Tg mice (22) was harvested, and RBC were lysed using RBC lysing reagent (Sigma-Aldrich, St. Louis, MO). Ten million GFP+ BM cells were injected i.v. into lethally irradiated (1100 rad) mice.
Flow cytometry and cell sorting
Single-cell suspensions were prepared from individual tissues by passage through a 100-µm pore size, nylon cell strainer (Falcon, BD Biosciences, Mountain View, CA) in RPMI medium containing 10% FCS. Tissue cell suspensions and PBMC cells were analyzed after lysing the RBC. Cells (105–106) were incubated with 5 µg/ml Fc block (BD PharMingen, San Diego, CA) and 300 µg/ml mouse IgG (Pierce, Rockford, IL). Cells were stained with Abs in 1x PBS, 1% BSA, and 0.1% sodium azide for 20 min at 4°C in the dark. mAbs to the following mouse surface markers were used: CD8 (53-6.7), pan NK (DX5), CD45R/B220 (RA3-6B2), CD11c (HL3), CD11b (M1/70), Ly6G/C (Gr-1, RB6-8C525-9-17), MHC II (IAb), CD3 (145-2C11), CD8b.2 (53-5.8), CD19 (1D3), CD86 (GL1), Ter119 (TER-119), CD4 (L3T4), CD90.2 (Thy1.2, 30-H12), and CD25 (IL-2Ra, PC61; all were purchased from BD PharMingen). F4/80 was purchased from Accurate Chemical and Scientific (Westbury, NY). To determine viability, samples were subsequently stained with 20 µl of 5 µg/ml propidium iodide (Calbiochem, La Jolla, CA). Events were acquired on a FACSCalibur (BD Biosciences) and analyzed using CellQuest software (BD Biosciences). For the identification and isolation of plasmacytoid cells, cells were stained with Abs against Ly6G/C, CD11c, and the lineage markers CD3, CD8b.2, CD11b, Ter119, and CD19. Cells were electronically gated on lineage-negative cells and screened for expression of CD11c and Ly6G/C. For isolation of plasmacytoid cells, CD11clowLy6G/Clow cells were sorted after electronic depletion of lineage-positive cells using a FACSVantage (BD Biosciences).
Mixed lymphocyte reaction
Allogeneic T and DC were isolated for an MLR. For the isolation of DC from non-Tg B6D2 mice, spleens were harvested, single-cell suspensions were prepared as described above, and cells were stained with Abs against CD11c and the lineage markers CD3, CD4, Thy1.2, IL-2R , B220, Gr-1, CD11b, F4/80, and Ter119. Lin- CD11c+ cells were sorted after electronic depletion of lineage-positive cells using a FACSVantage (BD Biosciences). For the isolation of DC from Tg mice (H2b/d), splenocytes were stained with Abs against CD11b, CD11c, and the lineage markers NK1.1, Thy1.2, Ter119, and B220. Cells were electronically gated on lineage-negative cells and screened for the expression of CD11c and CD11b. Lin- CD11chigh CD11blow/- and Lin- CD11chigh CD11b+ cells were sorted after electronic depletion of lineage-positive cells using a FACSVantage (BD Biosciences). Allogeneic CD4+ T cells were isolated from B10RIII (H2r) using Miltenyi MACS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Peripheral (axial, brachial, and inguinal) lymph nodes were harvested, and single-cell suspensions were prepared as described above. The cells were stained with biotinylated Abs against CD11b, CD8, B220, and IAd (MHC II), followed by incubation with streptavidin microbeads. CD4+ T cells were isolated to 95% purity by depleting the Ab-coated cells using a magnetic column according to the manufacturer's recommendations (Miltenyi Biotec).
The MLR reaction was performed in a 96-well, round-bottom tissue culture plate. Allogeneic CD4+ T cells (1x 105) were incubated with varying amounts of Tg DC (Lin- CD11chigh CD11blow/- and Lin- CD11chigh CD11b+) or control DC (CD11c) in 0.2 ml of DMEM containing penicillin/streptomycin, L-glutamine, 10% FCS, and 10-2 M 2-ME in humidified 10% CO2 in air for 5 days. The cultures were pulsed with 1 µCi of [3H]thymidine for 16 h, and the cells were harvested onto glass-fiber sheets for counting in a liquid scintillation counter (205 Betaplate; Wallac, Turku, Finland). Counts for the T cells, non-Tg DC, and Tg DC were between 200 and 300 cpm.
Cytospins
Spleens were harvested from control and Tg mice, single-cell suspensions were prepared, and Tg (CD11chigh CD11blow/- and Lin- CD11chigh CD11b+) and control (CD11c+) DC were sorted from total splenocytes as previously described. Cytospins were prepared by cytospinning 5 x 104 cells onto slides for 10 min at 500 rpm.
Viral stimulation of plasmacytoid cells
Sorted plasmacytoid cells were plated at 105 cells/well in 60 µl of RPMI medium containing penicillin/streptomycin, L-glutamine, and 10% FCS in a 96-well, round-bottom tissue culture plate. To stimulate IFN- production, 3 x 106 PFU of UV-inactivated HSV-1 virus was added to the plasmacytoid cells, and the incubation was continued for 24 h at 37°C in humidified 10% CO2 in air for 24 h. The culture supernatants were harvested, and the amount of IFN- was determined by ELISA as described below.
Statistical analysis
All experiments were performed in duplicate or triplicate, with three to five mice per group. The statistical difference between the mean values of groups was evaluated with Student's t test for comparisons between paired samples. Multiplicity was taken into account using the Bonferroni method. Results are presented as the mean ± SD. Data were analyzed using PRISM 3.0 (GraphPad, San Diego, CA).
Histology
Fresh mouse tissues were fixed immediately after necropsy in 10% buffered formalin and then processed for paraffin sections. Paraffin sections (5 µm) were prepared routinely and stained with H&E. For immunohistochemical staining, fresh-frozen sections were prepared and fixed with ice-cold acetone. Slides were then incubated with purified anti-CD11c (Chemicon International, Temecula, CA) for 1 h at room temperature, followed by incubation with biotinylated goat anti-hamster Ab (Vector Laboratories, Burlingame, CA) for 30 min. After incubation with an avidin-biotin-HRP complex (Vectastain Elite ABC kit, Vector Laboratories) for 30 min, the tissue sections were stained with NovaRed (Vector Laboratories) and counterstained with hematoxylin. For -gal histochemistry, fresh-frozen sections were fixed with 2% paraformaldehyde in PBS and then stained following a procedure previously described (23). Briefly, the slides were incubated overnight in the presence of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal; Roche, Indianapolis, IN) and counterstained with neutral red.
ELISA
PB was collected, and the serum was separated using Multivette 600 LH-Gel (Sarstedt, Nümbrecht, Germany). The serum was diluted 1/25, and the mFL concentration was determined using an ELISA kit specific for mFL (R&D Systems, Minneapolis, MN). The level of sensitivity of this assay is 5 pg/ml. Medium from the cultured cells was harvested 24 h after coculture with HSV. The IFN- concentration was determined using an ELISA kit specific for mIFN- (R&D Systems).
Doxycycline (DOX) preparation
For oral treatment, a 0.02 mg/ml solution of DOX (Sigma-Aldrich) was prepared by dissolving DOX in water containing 0.5% sucrose. The solution was placed in dark drinking water bottles and was changed every 4 days. For acute treatment, DOX (500 µg) was diluted in 0.2 ml of PBS and injected i.p.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate Tg mice that conditionally express mFL, we used the tetracycline-dependent gene expression system originally described by Gossen and Bujard (24). In this bigenic system the tet-activator protein (rtTA) is expressed constitutively from the activator transgene (Fig. 1). In the presence of tetracycline (or one of its analogs, such as DOX), the rtTA protein binds to a tetracycline-responsive promoter element present on a responder transgene and induces transgene expression. The generation of mice carrying and expressing the activator transgene in multiple tissues was previously described (19). Mice carrying the responder transgene were generated by microinjection of mouse eggs with the bidirectional transgene encoding -gal and mFL as described in Fig. 1.
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To select a Tg line for further studies we determined mFL serum levels in wild-type and four Tg lines before and after DOX treatment (500 µg i.p.). Serum levels of mFL in controls and Tg mice before DOX treatment averaged 0.5 ng/ml. Twenty-four hours after DOX injection the serum levels of mFL were substantially elevated (>50 ng/ml) in Tg animals from two of the four lines tested. FL levels in the serum of control mice treated with DOX did not differ from those in untreated control mice. The levels of mFL in the circulation were only marginally elevated (<2-fold) in the serum of the two remaining lines (Fig. 2). Significant differences in the expression levels among Tg lines is a common finding in many reports in the literature. It is possible that the lower levels of expression of mFL in two of the four lines may be caused by integration of the transgene into different chromosomal regions (positional effects). Tg mice from line 1372 (referred to hereafter as Tg mice), which showed the highest induction of mFL, were selected for expansion and further studies. Uninduced mice did not show any gross abnormalities and had a normal life span.
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As indicated in Fig. 1, the responder transgene encodes both mFL and -gal. To test whether DOX treatment induced -gal expression we treated Tg mice with DOX and examined the expression of -gal in several tissues, including spleen, muscle, thymus, pancreas, lymph nodes, brain, lung, heart, liver, kidney, intestine, trachea, and tongue. No -gal expression was detected before DOX treatment (Fig. 3, A, C, and E), but it could easily be detected in multiple tissues after DOX treatment. The most intense -gal staining was detected in the exocrine pancreas (Fig. 3F) and skeletal muscle (Fig. 3B). -Gal staining was also detected in kidney (Fig. 3D) and liver (not shown), but not in hemopoietic organs, such as BM, spleen, and thymus (not shown). The expression of -gal in the kidney was detected in the cortex. The likely source of transgene expression in this organ is the podocyte, since a recent study by Imai and coworkers (25) using the same promoter as that employed here demonstrated expression of GFP in this cell type. This expression pattern of -gal described here is similar to that observed previously using a different responder transgene, but the same activator line (19). Thus, the expression of the responder transgene in multiple tissues after DOX treatment is likely to account for the high levels of mFL in serum.
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It is well established that exogenous treatment of mice with hFL induces the expansion of DC in blood and several tissues (5, 10, 11). Therefore, having detected expression of the responder transgene in multiple tissues and high circulating levels of mFL, we examined the DC numbers in Tg mice. We first determined the number of DC in PB of animals treated with DOX. Acute treatment (96 h) induced dramatic changes in the circulating levels of mFL, which were, on the average, 200-fold higher than the values found in DOX-treated controls or nontreated Tg mice (Fig. 4A), but did not affect the number of CD11c+ cells in the circulation (Fig. 4B). However, chronic treatment of the Tg mice with DOX was associated with significant changes in the number of circulating DC. By 1 wk of DOX treatment, DC represented 40% of the cells in the PB. Interestingly, the high levels of mFL and the elevated number of DC in circulation persisted in treated animals throughout the course of the experiment (27 wk). There were no changes in the number of DC in the blood of nontreated Tg mice at any of the time points examined (data not shown). These results show that DOX treatment of Tg mice induces a dramatic elevation in the level of mFL and the number of DC in the circulation.
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40% of the PBL. DOX was then withdrawn, which led to a decrease in the level of mFL (data not shown) and in the relative number of DC in the circulation (Fig. 5). Within 5 wk of DOX removal, the number of DC had approached baseline levels. Interestingly, at this point, readministration of DOX to the Tg mice led to an increase in serum mFL levels and a blunted, but still significant, DC expansion. These results showed that it is possible to modulate the number of DC in the circulation by altering the concentration of DOX in the drinking water.
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Next we examined whether mFL expression would also lead to the expansion of DC in tissues. We examined tissue sections from untreated Tg mice and from Tg mice treated with DOX (0.02 mg/ml orally) for 6 wk. Analysis of H&E-stained paraffin sections showed a significant mononuclear cellular infiltrate in multiple tissues from Tg mice after DOX treatment (data not shown). These infiltrates, which were most abundant in lymphoid organs such as the spleen, lymph nodes, and thymus, were not associated with overt inflammation or necrosis. Phenotypic analysis of these infiltrates using immunohistochemistry indicated that the infiltrates consisted primarily of CD11c+ cells. In the thymus, CD11c+ cells could be found in both cortex and medulla (Fig. 6B). CD11c+ cells were also found in the portal region and the parenchyma of the liver (Fig. 6D), in the epithelium of the small and large intestines (Fig. 6F), in the pancreas (Fig. 6H), and in tongue striated muscle (Fig. 6J). With the exception of the thymus, where they accumulated in the medulla, CD11c+ cells were only rarely detected in similar tissues obtained from untreated Tg mice (Fig. 6, A, C, E, G, and I). Despite the elevated levels of mFL and the increased numbers of DC, there was no evidence of any abnormalities or autoimmune diseases; blood cell counts and blood chemistry parameters of hepatic, pancreatic, and renal function were normal.
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DC are frequently classified as lymphoid or myeloid based on their expression of the markers CD8 and CD11b, respectively. These DC populations apparently differ in their functions: lymphoid DC are less phagocytic, generally secrete IL-12, and initiate a Th1 response, whereas myeloid DC generally are more phagocytic and initiate a Th2 response (1). DC can also be classified on the basis of MHC class II expression, which distinguishes mature and immature DC. To further characterize the DC subsets present in lymphoid and hemopoietic tissues, we used flow cytometry. We observed dramatic changes in the number of DC in these tissues. In the spleen, for instance, there was a significant expansion of both immature (CD11c+IAb-/low) and mature (CD11c+IAb+) DC, and of myeloid or CD11c+CD8-CD11b+ and lymphoid or CD11c+CD8+CD11b- DC subsets (Fig. 7A). The CD11c+ IAb+ DC also expressed CD86 and varying levels of CD11b, but they did not express CD3, Gr-1, or Ter119 and were essentially negative for B220 (Fig. 7B). There was also a dramatic expansion in the relative (Table I) and absolute (Table II) numbers of various DC subsets in the thymus, mesenteric lymph node, BM, and PB. Most DC subsets were expanded in most tissues except the thymus, where primarily lymphoid (CD11c+CD8+CD11b-) DC were expanded. The apparent difference in cellularity of the thymus was not statistically significant between the controls and Tg mice treated with DOX.
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To determine whether Tg DC were functional, we sorted Lin- CD11chigh CD11blow/- and Lin- CD11chigh CD11b+ DC and tested them for the ability to stimulate allogeneic CD4+ T cells in an MLR assay. The stimulatory capacity of DC from Tg mice were compared with those of control (non-Tg) Lin- CD11c+ DC. DC from the Tg mice were as efficient as control DC in their ability to stimulate allogeneic T cells (Fig. 8).
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In humans, plasmacytoid DC are the major producers of IFN- in response to viruses or CpG stimulation (26, 27). Recently, the murine counterpart to the hIFN--producing plasmacytoid DC has been identified (28, 29). Plasmacytoid cells are CD11c+, B220+, and Ly6G/C+, are negative for CD19 and CD11b, and express low levels of MHC class II. Asselin-Paturel and coworkers (28) showed that these cells are lineage negative for Ter119, CD3, and CD8b.2. Therefore, to determine whether plasmacytoid cells were expanded in Tg mice after DOX treatment, we analyzed Ter119, CD8b.2, CD3, CD19, and CD11b lineage-negative PBL for the expression of Ly6G/C, CD11c, and B220. This DC subset constituted 5% of the lineage-negative cells before DOX treatment. After 7 wk of DOX treatment, the relative and absolute numbers of this cell population were increased in blood (Fig. 9A), BM, mesenteric lymph node, spleen, and thymus (Table III). A characteristic of both human (26) and murine (28, 29) plasmacytoid cells is that they secrete IFN- upon viral stimulation. To confirm that the Lin-Ly6G/ClowCD11clow cells in Tg mice were similar to the previously described plasmacytoid cells, we cultured them with UV-irradiated HSV for 24 h and measured IFN- in the culture supernatants. No IFN- could be detected in the supernatants of isolated cells in the absence of HSV. In contrast, IFN- could be readily measured in the supernatants of Lin-Ly6G/ClowCD11clow cells exposed to HSV (Fig. 9C). Only minimal expression (<10 pg/ml) of IFN- was detected in Ly6G/C-CD11c- cells cultured in the absence or the presence of HSV. These results conclusively demonstrate that in vivo expression of mFL can induce the expansion of a DC subset showing the characteristics of plasmacytoid cells.
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To determine whether the Tg mFL could drive DC proliferation from transplanted BM, we developed BM chimeras. These chimeras were generated by grafting BM from Tg mice expressing GFP in hemopoietic cells (22) into lethally irradiated mFL Tg mice. One week after BM transplantation, >95% of the PB cells of the host were GFP+. Treatment of these Tg mice with DOX caused a significant increase in the relative number of GFP+ DC in circulation. In nontreated mice GFP+ DC constituted 2% of all PBL, and in the treated mice they constituted 16% of the PBL (Fig. 10). These results indicate that expression of mFL by the host induced the expansion of DC precursors present in the grafted BM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Tg mice described here conditionally express mFL in multiple tissues. These animals were generated by intercrossing lines of mice carrying a responder transgene (encoding both FL and -gal) with a line of Tg mice expressing the trans-activator gene in a variety of organs (19). Animals carrying both transgenes were examined before and after DOX treatment, and a specific line (cross of activator line 72 with responder line 13) was chosen for the studies shown here. These animals were chosen because 1) we could not detect expression of mFL in serum or -gal in tissues before DOX treatment (e.g., there was no "leakiness" in transcription of the target genes); and 2) they showed a dramatic response to DOX treatment; the levels of mFL in circulation were rapidly and significantly elevated after a single DOX treatment. The elevated levels of mFL detected in the serum after DOX treatment were most likely caused by widespread expression of the transgene, as suggested by the pattern of -gal expression. In addition to -gal, the responder transgene used in our experiments encoded the natural transmembrane form of mFL, which can be processed to a soluble form (13). Because both the membrane-bound and soluble forms of mFL appear to be biologically active (14), either form may have contributed to the increased number of DC in multiple tissues.
Our results confirm and extend several reports in the literature regarding the ability of mFL to induce DC expansion in vivo (for review, see Ref. 31). Treatment of mice with hFL leads to the expansion of DC subsets in blood, BM, lymphoid tissues, liver, and lung (10, 11). These effects are probably mediated by the interaction of hFL with its tyrosine kinase receptor (flt3R) expressed by hemopoietic progenitor cells (9). In agreement with these studies, we observed a time-dependent increase in the number of DC in blood and lymphoid organs as a function of mFL expression. The CD11c+ MHCII+ spleen-derived DC showed similar phenotypic characterization as those reported previously in mice administered exogenous hFL (10). However, despite the significant increase in the relative and absolute numbers of various DC subsets, tissue cellularity was only slightly affected in our study (Table I), in contrast to the previous reports (5, 10, 32). The effective amount of mFL in the circulation may be the reason for the discrepancy. In previous studies exogenous hFL was used at high concentrations (10 µg/day), which could have led to higher systemic levels of hFL than those reported here. Unfortunately, a direct comparison between our studies is not possible because there is no information on the levels of circulating hFL or its half-life in the studies cited above. We should note that treatment of Tg mice with higher doses of DOX (2 mg/ml in food) leads to even higher levels of mFL and higher numbers of DC in PB and tissues (data not shown). Cessation of mFL induction led to normalization of DC numbers in blood. The number of DC gradually approached the baseline level after removal of DOX. Subsequent administration of DOX led to a second wave of DC expansion, indicating that the effect of mFL on DC expansion was reversible.
In our study the number of DC in PB remained elevated for an extended period of time (>9 mo), a finding in contrast to that reported by Brasel et al. (9) and Shaw et al. (32). These authors have observed a decline in the number of DC in mice treated for 3 wk with exogenous hFL and have suggested that this decline may have been caused by negative regulatory mechanisms or by a limited number of hemopoietic stem cells available for expansion (32). Our results do not support the concept that there is a limited number of hemopoietic stem cells available for expansion. Although the reasons for the discrepancy between our results and those described above remain unclear, we suggest that treatment of mice with hFL may have led to an immune response to this protein and subsequent inactivation of its biological activity.
The functionality of the expanded DC was determined by performing an MLR assay. We have shown that two independent populations of DC sorted from Tg mice are capable of stimulating the proliferation of allogeneic T cells as efficiently as DC isolated from control non-Tg mice. These data are consistent with those reported by Maraskovsky and coworkers (10), who found that similar populations of DC expressing high levels of CD11c and being CD11b+, CD11blow, or negative were capable of stimulating allogeneic or keyhole limpet hemocyanin-specific T cell proliferation.
Despite the significantly elevated levels of mFL and the increased number of DC, the Tg mice described here have not shown any abnormal phenotype. Previously, Juan and coworkers (33) reported that mice grafted with BM transduced with a retrovirus encoding mFL developed severe anemia, abnormal cellular infiltrates, vascular occlusion of the spleen, and premature death (33). Most deaths were observed 10–13 wk after transplantation. Our animals have now survived >36 wk of oral DOX treatment, and we have not observed any of those changes. There are, however, significant differences in our approaches. Juan and coworkers (33) used transduced BM cells in a transplant setting. The Tg mice reported here express mFL in most tissues, but not BM cells or spleen. The expression of mFL in the context of hemopoietic tissues could potentially favor the development of a severe phenotype. Since the levels of mFL were not measured in that study, it remains possible that they were in vast excess over what was observed here. Thus, the conditional Tg system described here provides a method for continued expansion of DC for extended periods of time without deleterious effects on the health or survival of these mice. The system described here will be particularly useful for obtaining DC from PB without sacrificing the mice.
One of the most important findings of our study was the demonstration that mFL expression can promote the expansion of plasmacytoid cells. Plasmacytoid cells or DC precursors are the primary cells producing IFN- in the blood (26) and have a major impact on both innate and adaptive immune responses. The production of IFN- by plasmacytoid cells leads to the mobilization and activation of macrophages and NK cells during the acute innate response (1, 26, 34, 35). Subsequently, these plasmacytoid cells differentiate and mature into DC, which stimulate T cells to differentiate into Th1 or Th2 subsets or into T cells that produce both IFN- and IL-10 (for review, see Refs. 1 and 30).
Recently, the murine counterpart of human plasmacytoid cells was identified (28, 29). In contrast to human plasmacytoid cells, which lack expression of CD11c (26), murine plasmacytoid cells express CD11c. These murine cells also express B220 and Ly6G/C and minimal levels of MHC class II, CD19, and CD11b, but are lineage negative for Ter119, CD3, and CD8b.2 (28). Here we show a dramatic expansion in PB and tissues of DOX-treated mice of a population of cells that is lineage negative (Ter119, CD8b.2, CD3, CD19, and CD11b), but expresses Ly6G/C, CD11c, and B220. Consistent with these cells being plasmacytoid cells, we detected a significant increase in IFN- production upon exposure of these isolated cells to UV-inactivated HSV. IL-12 (p40) was also measured in the supernatants of plasmacytoid DC (pDC) cultures (data not shown). Similar to what was found by Gilliet and coworkers (36), we could not detect IL-12 in the absence of virus. However, we could not document the modest up-regulation of IL-12 by pDCs after viral stimulation as reported by those authors. The reasons for this discrepancy are not clear, but may stem from the source of the DC. We obtained our pDC from the spleens of mice chronically exposed in vivo to mFL, whereas Gilliet et al. (36) obtained pDC from mBM by stimulation in vitro with hFL. In both our report and that by Gilliet, however, there was irrefutable production of IFN- by pDCs upon HSV-1 incubation. These results indicate that mFL expression in vivo can lead to the expansion of a DC subset with the characteristics of murine plasmacytoid cells in blood and multiple tissues. Thus, the Tg mice described here should aid in the characterization of this newly defined murine DC subtype.
Finally, the Tg mice described here should prove very useful for the analysis of trafficking and localization patterns of DC. First by providing the source of a large number of DC of a given subtype that can be isolated and subsequently labeled and used in adoptive transfer studies, and second, by serving as a host for the expansion of genetically modified DC. We have shown here that grafting the FL-expressing mice with BM from GFP-expressing mice can induce the expansion of grafted DC. We anticipate that this approach will be particularly useful in defining the roles of specific molecules, such as chemokines, in DC trafficking. Chemokines induce the chemotaxis of DC in vitro, and chemokine receptors are differentially expressed by DC subsets (reviewed in Refs.1 and37, 38, 39). Targeted deletion of the chemokine receptor CCR6 abolishes the ability of a specific subset of DC (CD11b+CD11c+) to localize within the subepithelial dome of Peyer's patches (40), and inactivation of CCR7 reduces the migration of mature DC from the skin to the draining lymph nodes (41). These results strongly suggest that chemokines are important regulators of DC trafficking in vivo and that the migration of specific subsets may be controlled by specific chemokines. Experiments involving the transfer of GFP+ BM cells from mice lacking a particular chemokine receptor into lethally irradiated Tg mice should help elucidate the role of this chemokine receptor in DC trafficking and localization.
In summary, we describe a novel method to generate a large number of mDC in vivo. The method described here is based on the induction of Tg mFL in multiple tissues by DOX. The method is noninvasive and reversible, does not require frequent manipulation of the mice, and is not associated with overt pathology, even after prolonged treatment with DOX. DOX treatment of Tg mice allows for the expansion of all DC subsets analyzed and for obtaining large numbers of DC from blood and tissues. This method should thus greatly facilitate gene expression profiling and trafficking studies of DC subsets and contribute to a better understanding of the role of DC in health and disease.
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Acknowledgments |
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-actin promoter cassette. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Sergio A. Lira, Mount Sinai School of Medicine, Immunobiology Center, 1425 Madison Avenue, Box 1630, New York, NY 10029-6574. E-mail: sergio.lira{at}mssm.edu
3 Abbreviations used in this paper: DC, dendritic cells; BM, bone marrow; DOX, doxycycline; FL, Flt3 ligand; -gal, -galactosidase; GFP, green fluorescent protein; h, human; m, murine; PB, peripheral blood; Tg, transgenic; pDC, plasmacytoid DC.
Received for publication February 25, 2002. Accepted for publication January 6, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-interferons: 40th anniversary of the discovery of interferons. Cancer Res. 58:2489.This article has been cited by other articles:
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  D. Kingston, M. A. Schmid, N. Onai, A. Obata-Onai, D. Baumjohann, and M. G. Manz The concerted action of GM-CSF and Flt3-ligand on in vivo dendritic cell homeostasis Blood, July 23, 2009; 114(4): 835 - 843. [Abstract] [Full Text] [PDF]
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 A. P. Martin, S. Rankin, S. Pitchford, I. F. Charo, G. C. Furtado, and S. A. Lira Increased Expression of CCL2 in Insulin-Producing Cells of Transgenic Mice Promotes Mobilization of Myeloid Cells From the Bone Marrow, Marked Insulitis, and Diabetes Diabetes, November 1, 2008; 57(11): 3025 - 3033. [Abstract] [Full Text] [PDF]
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Y. Huang, I. J. Fugier-Vivier, T. Miller, M. J. Elliott, H. Xu, L. D. Bozulic, P. M. Chilton, and S. T. Ildstad Plasmacytoid Precursor Dendritic Cells From NOD Mice Exhibit Impaired Function: Are They a Component of Diabetes Pathogenesis? Diabetes, September 1, 2008; 57(9): 2360 - 2370. [Abstract] [Full Text] [PDF]
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 G. C. Furtado, B. Pina, F. Tacke, S. Gaupp, N. van Rooijen, T. M. Moran, G. J. Randolph, R. M. Ransohoff, S. W. Chensue, C. S. Raine, et al. A Novel Model of Demyelinating Encephalomyelitis Induced by Monocytes and Dendritic Cells J. Immunol., November 15, 2006; 177(10): 6871 - 6879. [Abstract] [Full Text] [PDF]
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E. Bockamp, C. Antunes, M. Maringer, R. Heck, K. Presser, S. Beilke, S. Ohngemach, R. Alt, M. Cross, R. Sprengel, et al. Tetracycline-controlled transgenic targeting from the SCL locus directs conditional expression to erythrocytes, megakaryocytes, granulocytes, and c-kit-expressing lineage-negative hematopoietic cells Blood, September 1, 2006; 108(5): 1533 - 1541. [Abstract] [Full Text] [PDF]
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R. Pelayo, J. Hirose, J. Huang, K. P. Garrett, A. Delogu, M. Busslinger, and P. W. Kincade Derivation of 2 categories of plasmacytoid dendritic cells in murine bone marrow Blood, June 1, 2005; 105(11): 4407 - 4415. [Abstract] [Full Text] [PDF]
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T. Tamura, P. Tailor, K. Yamaoka, H. J. Kong, H. Tsujimura, J. J. O'Shea, H. Singh, and K. Ozato IFN Regulatory Factor-4 and -8 Govern Dendritic Cell Subset Development and Their Functional Diversity J. Immunol., March 1, 2005; 174(5): 2573 - 2581. [Abstract] [Full Text] [PDF]
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M. J. Osborn, P. L. Ryan, N. Kirchhof, A. Panoskaltsis-Mortari, F. Mortari, and K.-S. R. S. Tudor Overexpression of murine TSLP impairs lymphopoiesis and myelopoiesis Blood, February 1, 2004; 103(3): 843 - 851. [Abstract] [Full Text] [PDF]
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C. Asselin-Paturel, G. Brizard, J.-J. Pin, F. Briere, and G. Trinchieri Mouse Strain Differences in Plasmacytoid Dendritic Cell Frequency and Function Revealed by a Novel Monoclonal Antibody J. Immunol., December 15, 2003; 171(12): 6466 - 6477. [Abstract] [Full Text] [PDF]
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, control; 
, Tg mice treated with DOX. *, p < 0.001 for DOX-treated Tg vs no treatment (0); **, p < 0.01 for DOX-treated Tg at 14 and 16 wk vs Tg at 9 wk or no treatment.
