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Departments of
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Pathology and
Cell Biology and Physiology, University of New Mexico, Albuquerque, NM 87131
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the lung, DCs reside within and beneath airway epithelium, in alveolar septa, in the connective tissue surrounding pulmonary veins and airway vessels, and in the lung vascular compartment (8, 9, 10, 11). The regulation of immune responses in the lung is a complex process that involves maintaining tolerance to endogenous self-Ags and innocuous inhaled agents, while retaining the capacity to mount immune responses against invading microorganisms. DCs take up Ags, whether self or non-self and readily degrade them to produce antigenic peptides that bind to MHC molecules for recognition by TCRs on T cells (11, 12, 13, 14, 15, 16, 17, 18, 19). Ag binding to Toll-like receptors (TLRs) or endocytic receptors may also signal DCs to mature and produce cytokines that contribute to the polarization of Th cells (20, 21, 22, 23, 24, 25, 26). We and others propose that DCs play a pivotal role in inducing naive T cells to become tolerogenic or immunogenic, depending on their maturation status (expression of MHC class II and accessory molecules) and cytokine response as a result of Ag binding (20, 21, 22, 23, 27, 28, 29). In addition, the differential expression of TLRs and endocytic receptors may bias the functional abilities of the DC subset(s) (24, 25, 26).
A limitation in studying lung DCs is that they represent only 1–2% of total lung cells in the absence of ongoing inflammation and immune responses. Thus, we sought out the reagent, FMS-like tyrosine kinase 3 ligand (Flt3L) that was previously reported to increase the number of DCs in various organs, including the lungs of mice (2, 30) and assessed whether phenotype and functional differences exist between vehicle and Flt3L-treated lung DCs. Flt3L is a hemopoietic growth factor whose receptor, CD135, is a member of the type III receptor tyrosine kinase family. Maraskovsky et al. (2) showed that in the lungs of FLt3L-treated mice, the relative numbers of both CD11b-positive and CD11b-negative DCs within the CD205-positive population were increased. Shurin et al. (31) showed that Flt3L-induced spleen DCs were morphologically and phenotypically indistinguishable from DCs obtained from control mice, but exhibited more potent APC activity in a MLR in vitro and in initiating antitumor responses in vivo. In the spleen, Flt3L expanded both the CD11c-positive, CD11b-positive myeloid, and CD11c-positive, CD11b-negative lymphoid DC subsets (2, 6, 31). Taken together, these studies suggest that Flt3L may alter the proportion and function of DC subsets in the lungs of treated mice.
In the present study, we investigated the effects of in vivo Flt3L administration on the generation, phenotype, and function of lung DCs to evaluate whether Flt3L favors the expansion and maturation of a particular DC subset. This study extends the limited study on Flt3L-induced lung DCs conducted by Maraskovsky et al. (30) and demonstrates that Flt3L injections results in an increased number of lung DCs, preferentially of the myeloid subset that are functionally more active and phenotypically more mature than vehicle-treated lung DCs. Our data provide the first thorough characterization of lung DCs generated in vivo with Flt3L. Our data support a possible use for Flt3L as part of an immunotherapy protocol to enhance immunity to infectious agents and tumors that target the lung, because Flt3L favors the expansion and partial maturation of lung DCs of the myeloid subset.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c (I-Ad MHC class II) mice were purchased from the National Cancer Institute (Frederick, MD). BALB/c mice were injected (100 µl) i.p. once daily with either mouse serum albumin (MSA; 0.01% in PBS) or Flt3L (10 µg in 0.01% MSA) for 9 consecutive days. DO11.10 OVA-TCR-transgenic (Tg) heterozygotes were bred at the University of New Mexico Animal Resources Facility (Albuquerque, NM) by breeding OVA-TCR Tg heterozygous mice (a kind gift from Dr. D. Loh (32) with BALB/c female mice. T lymphocytes from DO11.10 mice express a TCR that is specific for the OVA323–339 peptide fragment, in the context of I-Ad. All mice were housed under specific pathogen-free conditions and used between 10 and 12 wk of age. The University of New Mexico Animal Resources Facility is accredited by the American Association for Accreditation of Laboratory Animal Care, and all animal protocols were reviewed and approved by the University of New Mexico Institutional Animal Care and Use Committee.
Reagents
Flt3L was a kind gift from Dr. S. Lyman (Amgen, Seattle, WA). MSA was purchased from Sigma-Aldrich (St. Louis, MO). The OVA323–339 peptide was synthesized by Research Genetics (Huntsville, AL). All cells were cultured in cRPMI, which is defined as RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME, and 10% heat-inactivated FBS (Life Technologies, Grand Island, NY).
Immunohistochemistry
Lungs were inflated with a mixture of 25% Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) in 1x PBS and subsequently frozen in liquid nitrogen and stored at -70°C until sectioned. Seven-micrometer frozen sections were mounted on precleaned superfrost plus micro slides (VWR Scientific, West Chester, PA), dried at room temperature for 1 h, and then fixed in cold acetone for 10 min. Sections were washed in TBS (0.05 M Tris-HCl (pH 7.6), 0.3 M NaCl, and 0.1% Tween 20), then treated for 10 min with a commercial peroxidase block (DAKO, Carpinteria, CA) and incubated for 30 min with a protein-blocking reagent (DAKO) containing 10% normal mouse serum (Jackson ImmunoResearch Laboratories, West Grove, PA). A commercial biotin-blocking kit (DAKO) was then used according to the manufacturer’s instructions. Biotinylated anti-CD11c (HL3, hamster IgG), anti-CD45R/B220 (RA3-6B2, rat IgG2a), anti-I-Ad/I-Ed (2G9, rat IgG2a), hamster IgG1 (G235-2356), and rat IgG2a (R35-95) were purchased from BD PharMingen (San Diego, CA) and diluted to working concentrations in Ab diluent containing background reducing components (DAKO). The isotype control Abs were diluted to the same concentration as their respective specific Ab. Sections were incubated overnight with Abs at 4°C in a humidified chamber. The next day the sections were incubated for 1 h at room temperature with an avidin-biotin-HRP conjugate (ABC reagent; Vector Laboratories, Burlingame, CA) and then washed with TBS. Immunoreactivity was detected using a diaminobenzidine substrate (Vector Laboratories). Sections were counterstained with methyl green. H&E staining was done by TriCore Reference Laboratories (Albuquerque, NM).
Preparation of low-density and pulsed low-density lung cells
BALB/c lung cells were prepared as described previously (13, 14). Briefly, mice were injected i.p. with 150 U heparin (ICN Biomedicals, Aurora, OH) and the anesthetic Avertin (44 µM 2,2,2-tribromoethanol, 71 µM tert-amyl alcohol; Aldrich, Milwaukee, WI) in distilled water and exsanguinated. Mice lung airways were lavaged using an intratracheal cannula with cold PBS containing 0.6 mM EDTA. The pulmonary vasculature was perfused with sterile saline to remove peripheral blood cells. The lavaged, perfused lungs were minced, and enzyme treated at 37°C for 90 min in cRPMI containing collagenase A (0.7 mg/ml; Boehringer Mannheim, Indianapolis, IN) and type IV bovine pancreatic DNase I (30 µg/ml; Sigma-Aldrich). Digested lung tissue was tapped through a wire screen, particulate matter was removed by rapid filtration through a nylon wool plug, and the filtered cells were washed twice in HBSS (Life Technologies). Lung cells were counted using a hemocytometer and cytospins were made for cell differential analysis. Lung cells were resuspended in high-density Percoll (Pharmacia, Piscataway, NJ; 
= 1.075 g/ml), overlaid with an equal volume of lower density Percoll ( = 1.030 g/ml), and centrifuged at 400 x g for 20 min. Cells at the 1.075/1.030 Percoll interface, which are enriched for mononuclear cells were recovered and washed with HBSS, are referred to as "low-density lung cells." For Ag presentation experiments, low-density lung cells were resuspended (2–3 x 106 cells/ml) in cRPMI with or without OVA323–339 peptide (10 µM) in 50 ml of polypropylene conical centrifuge tubes at 37°C in 5% CO2 for 18–24 h. Thereafter, peptide-pulsed or mock-pulsed lung DCs were sorted by flow cytometry, as described below in "Isolation of Pulsed Lung DCs." Low-density lung cells were pulsed within 3 h after the lung tissue harvest.
Four-color immunophenotyping of DCs
Low-density lung cells or peptide-pulsed low-density lung cells were first incubated with purified anti-CD16/CD32 (2.4G2, rat IgG2b; BD PharMingen) to block both FcR-mediated binding of staining Ab and cell aggregation by Fc
RII binding by macrophages of Ab-stained DCs and B cells. Subsequently, cells were stained with anti-CD11c-FITC (HL3, hamster IgG), anti-CD45R/B220-PerCP (RA3-6B2, rat IgG2a), and biotinylated anti-I-Ad/I-Ed (2G9, rat IgG2a) and a PE-conjugated mAb to the surface molecule of interest (anti-CD86 (GL1, rat IgG2a), anti-CD80 (16-10A1, hamster IgG), anti-CD40 (3/23, rat IgG2a), anti-CD24 (M1/69, rat IgG2b), anti-CD11a (2D7, rat IgG2a), anti-CD54 (3E2, hamster IgG), anti-CD25
(3C7, rat IgG2b), anti-CD1d (1B1, rat IgG2b), anti-CD8 (53-6.7), anti-CD11b (M1/70, rat IgG2b), rat IgG2a (R35-95), rat IgG2b (R35-38), and hamster IgG (G235-2356). I-Ad binding was identified by incubation with streptavidin-allophycocyanin. All primary and secondary reagents were purchased from BD PharMingen. Cells were stained at 4°C and washed three times with PBS containing 2% FBS and 40 µg/ml EDTA after each incubation step. Cells were fixed with 2% paraformaldehyde. A BD Biosciences FACSCalibur (Mountain View, CA) was used for data acquisition and WinList (Verity Software House, Topsham, ME) software was used for analysis.
Isolation of pulsed lung DCs
For the isolation of overnight peptide-pulsed or mock-pulsed DCs from the lung, peptide-pulsed low-density lung cells were first incubated with anti-CD16/CD32 and then stained with anti-CD11c-FITC, -CD3-PE (17A2, rat IgG2b; BD PharMingen), -CD45R/B220-PerCP, and biotinylated anti-I-Ad/I-Ed. I-Ad binding was identified by secondary staining with streptavidin-Red 613 (Sigma-Aldrich). To obtain DCs in high purity, cells expressing both CD11c and I-Ad, but negative for CD3 and CD45R/B220, were collected in a tube using a MoFlo cytometer (Cytomation, Fort Collins, CO). The forward scatter/side scatter profile of stained cells was used to gate out debris. A second gate was set on cells expressing CD11c, but lacking expression of CD3 to exclude T lymphocytes. A third gate was set on cells expressing I-Ad, but lacking expression of CD45R/B220 to exclude B lymphocytes and plasmacytoid DCs. All three gates were used together to sort lung DCs. Sorted DCs were 99% viable, determined by trypan blue dye exclusion.
Isolation of naive CD4-positive DO11.10 T cells
To prepare T cells for sorting, spleen cells from DO11.10 OVA-TCR Tg heterozygous mice were passed on nylon wool, incubated with anti-CD16/32, and then stained with FITC-conjugated anti-CD4 (RM4-5, rat IgG2a), PE-conjugated anti-CD62L (MEL-14, rat IgG2a), and biotinylated anti-I-Ad/I-Ed, followed by secondary staining with streptavidin-PerCP. To obtain naive CD4-positive T cells in high purity, cells expressing both CD4 and CD62 ligand (CD62L), but negative for I-Ad, were collected in a tube. The forward scatter/side scatter profile of stained cells was used to set a gate on the lymphocyte population. A second gate was set on cells expressing high levels of CD4 and CD62L. A third gate was set on cells expressing high levels of CD62L, but lacking expression of I-Ad to exclude APCs. All three gates were used together to sort naive (CD62L-positive) CD4-positive T cells using a MoFlo cytometer. Sorted cells were 99% viable as determined by trypan blue dye exclusion. The presence of I-Ad-expressing cells in the sort was determined by culturing sorted T cells with OVA323–339 peptide and quantifying lymphoproliferation based on [3H]thymidine incorporation.
Ag-specific T cell proliferation
Sorted OVA323–339 peptide-pulsed or mock-pulsed lung DCs were cultured with sorted naive CD4-positive DO11.10 T cells (2.5 x 104/well) in 200 µl of cRPMI, in triplicate, in 96-well flat-bottom plates at 37°C in 5% CO2 for 4 days. Eighteen hours before harvesting, 0.5 µCi [methyl-3H]thymidine (Amersham, Arlington Heights, IL) was added to each well. Controls included each cell type with medium only and medium with 10 µM OVA323–339 peptide where appropriate.
Statistics
All ANOVA models and unpaired t tests were performed with the Statview software (SAS Institute, Cary, NJ). A p 
0.05 was considered to be significant.
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Results |
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Studies were initiated to confirm the findings of Maraskovsky et al. (2) that described an increase in nucleated cell numbers in the lungs of Flt3L-treated mice by evaluating the total leukocyte count and the absolute cell differential numbers of lungs cells from mice pretreated with Flt3L. Enzyme-digested lung cells from Flt3L- and vehicle-treated mice were counted and analyzed by cell differentials. Intraperitoneal administration of Flt3L to pathogen-free mice for 9 consecutive days resulted in a significant increase in total leukocytes in the lung as compared with vehicle-treated mice (mean cell count ± SE: Flt3L, 42.19 ± 4.35 x 106; vehicle, 12.54 ± 1.87 x 106; p < 0.0001; n = 8). Flt3L-pretreated mice demonstrated a significant increase in the absolute numbers of granulocytes (Flt3L, 6.92 ± 1.11 x 106; vehicle, 1.85 ± 0.17 x 106; p = 0.0005; n = 8), lymphocytes (Flt3L, 17.24 ± 2.04 x 106; vehicle, 9.20 ± 1.84 x 106; p = 0.0110; n = 8), and monocytoid cells (Flt3L, 18.08 ± 2.14 x 106; vehicle, 1.50 ± 0.09 x 106; p < 0.0001; n = 8). Flt3L elicited, on average, a 3.7-fold increase in granulocytes, a 1.9-fold increase in lymphocytes, and a 12.1-fold increase in monocytoid cells. The total leukocyte counts and differentials of vehicle-treated mice were similar to those of untreated mice (n = 5).
Flt3L treatment increased the number of lung cells expressing CD11c, MHC class II, and CD45R/B220
We analyzed lung cells in tissue sections from Flt3L-treated mice for localization, cellular content, and expression of cellular CD11c, MHC class II (I-Ad), and CD45R/B220 (Fig. 1). CD45R/B220 is a surface marker commonly found expressed on B cells, but is not restricted to the B cell lineage. Others have described a subset of CD11c-positive, MHC class II-positive DCs that express CD45R/B220 and proposed that they are the murine equivalent of human plasmacytoid DCs (4, 33). Lung cells expressing CD11c, MHC class II, and CD45R/B220 were present beneath the airway epithelium, in alveolar septa, and in connective tissue surrounding the pulmonary veins and airway vessels in both Flt3L- and vehicle-treated mice. Hypercellularity was seen in the lungs of mice treated with Flt3L, consistent with the increased absolute lung cell count. Based on a qualitative analysis of immunohistochemistry staining, this hypercellularity is likely due to an increased number of cells expressing the surface markers CD11c, MHC class II, and CD45R/B220. The increase was most striking in alveolar septa rather than in and beneath bronchiolar epithelium.
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Enzyme-digested low-density lung cells from Flt3L- and vehicle-treated mice were analyzed by immunofluorescent labeling and four-color flow cytometry to determine the presence and heterogeneity of DCs and the level of expression of MHC class II on these cells (Fig. 2). Based on expression of CD11c and CD45R/B220, at least three subsets of DCs were distinguished in Flt3L-treated mice. One CD11c-positive subset did not express CD45R/B220 (Fig. 2A, R1), while the other two subsets expressed varying levels of both CD11c and CD45R/B220 (Fig. 2A, R2 and R3). In vehicle-treated mice, the CD11c-positive CD45R/B220-negative subset was clearly present (Fig. 2B, R1), whereas the CD11c-positve CD45R/B220-positive DC subsets were not as easily distinguishable, due to low event counts (Fig. 2B, R2 and R3). In both Flt3L- and vehicle-treated mice, we were unable to further characterize the CD45R/B220-positive DC subsets because of a combination of high background binding of the isotype controls and autofluorescence. Therefore, we focused the analysis on the CD11c-positive, CD45R/B220-negative DC subset, the predominate subset in the lungs of both Flt3L- and vehicle-treated mice.
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19-fold greater than vehicle-treated mice (0.16 ± 0.03 x 106, Table I). Vehicle-treated mice and untreated mice showed similar numbers of DCs (data not shown).
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We analyzed the CD11c-positive, CD45R/B220-negative, MHC class II-positive lung DCs that were CD11b positive and CD11b negative. We determined that the CD11b-positive myeloid subset was preferentially expanded in the lungs of Flt3L-treated mice over the CD11b-negative lymphoid subset (Figs. 2, C and D, and 3 and Table II). Myeloid DCs in the CD11c-positive, CD45R/B220-negative, MHC class II-positive population were significantly greater in mice treated with Flt3L (89.0 ± 2%) than in vehicle-treated mice (21.0 ± 2%). The absolute number of myeloid lung DCs from Flt3L-treated mice (2.71 ± 0.25 x 106) showed approximately a 90-fold increase as compared with vehicle-treated mice (0.03 ± 0.005 x 106). In contrast, the absolute number of lymphoid DCs from Flt3L-treated mice (0.37 ± 0.15 x 106) was only 3-fold increased as compared with vehicle-treated mice (0.12 ± 0.02 x 106, Table I).
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Our previous studies showed that DCs from the lungs of pathogen-free mice express and use multiple accessory molecules in their role as initiators of primary T cell responses, including CD86, CD80, CD11a, and CD54 (13, 14). DCs from Flt3L- and vehicle-treated mice were analyzed by immunofluorescent labeling and flow cytometry to determine whether they varied in the expression (percent positive and mean fluorescence intensity) of a number of different accessory molecules (Fig. 3 and Table II). The CD11c-positive, CD45R/B220-negative, MHC class II-positive DC population from Flt3L-treated mice demonstrated a significantly higher percentage of cells expressing CD86, CD40, CD24, CD1d, CD8, and CD11b (as noted earlier) as compared with vehicle-treated mice. In contrast, no difference in the percent positive cells expressing CD80, CD11a, CD54, and CD25 was distinguished between Flt3L- and vehicle-treated lung DCs. Interestingly, of the positive cells, the mean fluorescence intensity of CD86, CD80, and CD1d was significantly higher on vehicle-treated lung DCs. Vehicle-treated mice and untreated mice showed similar expression of accessory molecules (data not shown).
Our previous studies have shown that lung DCs increase expression of a number of selected accessory molecules during an overnight lung suspension culture (13). To determine whether Flt3L- and vehicle-treated lung DCs also have this capacity, we evaluated the phenotypes of CD11c-positive CD45R/B220-negative MHC class II-positive DCs from Flt3L- and vehicle-treated mice held overnight in lung cell suspension cultures. The expression of MHC class II on Flt3L-treated DCs remained unchanged after overnight culture, whereas an increase in both the percent positive and mean fluorescence intensity was seen with vehicle-treated DCs (data not shown). In contrast, the percentage of cells expressing CD86, CD80, CD40, and CD25 increased for both cultured Flt3L- and vehicle-treated DCs as compared with freshly isolated DC counterparts. Cultured Flt3L-treated DCs showed the highest percentage of positive cells for each marker that was increased.
Flt3L treatment enhanced the capacity of lung DCs to stimulate naive T cells in vitro
To determine whether these differences in MHC class II and accessory molecule expression by freshly isolated Flt3L- and vehicle-treated lung DCs made these populations functionally different, we compared their ability to present OVA323–339 peptide to naive CD4-expressing T cells isolated from DO11.10 TCR Tg mice. Freshly isolated low-density lung cells from Flt3L- and vehicle-treated mice were cultured in suspension with or without OVA323–339 peptide overnight. The next day the peptide-pulsed or mock-pulsed cells were extensively washed and CD11c-positive, CD45R/B220-negative, MHC class II-positive lung DCs were isolated and added in graded numbers to naive CD4-positive DO11.10 T cells. As shown in Fig. 4, Flt3L-treated DCs were significantly more efficient than vehicle-treated DCs in stimulating naive Ag-specific Th cell proliferation at all concentrations of DCs tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four CD11c-positive DC subsets were detected in the lungs of both FLt3L- and vehicle-treated mice. The first subset expressed CD11b and lacked expression of CD45R/B220, attributes of myeloid DCs. The second subset lacked expression of both CD11b and CD45R/B220, characteristic of lymphoid DCs. The third and fourth subsets were minor populations that expressed varying levels of both CD11c and CD45R/B220 and are considered plasmacytoid DCs. We were unable to further characterize these minor DC subsets because of high background binding of the isotype controls to these cells. FACS analysis allowed us to quantify a 28-fold increase of the absolute numbers of CD11c-positive nonplasmacytoid DCs in the lungs of Flt3L-treated mice over vehicle-treated mice. Further analysis using the marker CD11b revealed a 90-fold increase in the absolute number of myeloid lung DCs from Flt3L-treated mice over vehicle-treated mice. In contrast, the absolute number of lymphoid lung DCs from Flt3L-treated mice showed only a 3-fold increase. The observation that Flt3L induced both myeloid and lymphoid lung DCs was expected, because previous reports in the spleen and the gut-associated lymphoid tissue described an increase in both DC subsets in response to Flt3L injections (2, 6, 7, 31, 34). However, it was a surprise that within the heterogeneity of lung DCs in Flt3L-treated mice, the myeloid subset prevailed so dramatically over the lymphoid subset, whereas in vehicle-treated mice, the opposite was observed. The dominance of lymphoid DCs in the lungs of vehicle-treated mice is in agreement with Dodge et al. (35) who described low to negative expression of CD11b on pulmonary DCs from BALB/c mice.
In the current study, we asked whether APC functional differences between Flt3L-treated and vehicle-treated nonplasmacytoid lung DCs might be attributable to differences in the expression of the required combination of peptide-MHC complexes and accessory molecules necessary to engage the TCR and initiate T cell proliferation. Based on the phenotypic data, Flt3L treatment not only increased the percentage of nonplasmacytoid lung DCs expressing MHC class II, but also the level of expression of this molecule, as compared with vehicle-treated lung DCs. A higher percentage of Flt3L-teated lung DCs expressed the accessory molecules CD86 and CD40, known to be expressed on mature DCs and to interact with counterreceptors on T cells to enhance costimulation and adhesion. The percentage of lung DCs from Flt3L-treated mice expressing CD80 was similar to that in vehicle-treated mice. This observation that the percentage of CD80 was not different was surprising, because CD80 is highly expressed on mature lung DCs (13, 14). These observations suggest that Flt3L-treated lung DCs are more differentiated than vehicle-treated lung DCs, but have not yet reached full maturation. In fact, Flt3L-treated lung DCs matured further in accessory molecule expression under culture conditions previously shown to differentiate lung (13).
Recent studies have suggested that CD8 is a maturation marker that represents the last stage of DC differentiation (36, 37). However, our previous data with untreated mice (13) and our current data with Flt3L- and vehicle-treated mice showed that after an overnight culture the expression of CD8 did not increase. We speculate that the overnight culture conditions used to mature DCs were not sufficient to cause full differentiation and up-regulation of CD8. It is possible maturation conditions that signal through TLRs expressed on DCs will induce full maturation and CD8 expression. Others (36) have associated the differentiation process with up-regulation of both CD24 and CD8, but we see similar high expression of CD24 on both semimature Flt3L- and immature vehicle-treated lung DCs.
Data are accumulating that suggests the immunologic outcome, whether induction of tolerance or active immunity following expansion of DCs at mucosal sites may be a consequence of the state of maturation of the mobilized DCs. For example, Flt3L-mobilized gut-associated lymphoid tissue DCs enhanced the induction of tolerance induced by oral administration of soluble protein. However, tolerance was abrogated by additionally treating these mice with a potent adjuvant or the inflammatory cytokine IL-1 (34, 38). The level of TLR signaling may also determine the type of immunogenic response generated in addition to the proposed correlation of DC maturation status with immunologic outcome (39, 40). For example, studies have shown that intranasal sensitization of mice with Ag containing low or high levels of LPS induced Th2 or Th1 immune responses, respectively (39). We are currently testing the hypothesis that Flt3L-treated lung DCs instilled into the lungs will induce tolerance to inhaled soluble proteins unless an inflammatory signal is coadministered and that the degree of signaling through TLRs will determined whether a Th1-or Th2-type immune response ensues. We also are asking whether the immunologic consequences of DC mobilization to the lung may be skewed by the specific subset(s) of DCs that are expanded; i.e., lymphoid vs myeloid.
In summary, Flt3L injections resulted in an increased number of lung DCs, preferentially of the myeloid subset, that were functionally more active and phenotypically more mature than vehicle-treated lung DCs. Mobilizing lung DCs with Flt3L may provide an opportunity to elicit diverse immunological outcomes in different clinical settings. The prospect of using Flt3L to augment vaccine responses against pathogenic lung organisms, promote antitumor responses, or perhaps induce tolerance or dampen an allergic response in the lung holds sufficient promise that additional studies are warranted.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cell; Flt3L, FMS-like tyrosine kinase 3 ligand; TLR, Toll-like receptor; MSA, mouse serum albumin; Tg, transgenic; CD62L, CD62 ligand.
Received for publication June 16, 2003. Accepted for publication January 23, 2004.
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+ dendritic cells originate from the CD8- dendritic cell subset by a maturation process involving CD8, DEC-205, and CD24 up-regulation. Blood 99:999.-positive dendritic cells from a common myeloid progenitor. Science 290:2152.This article has been cited by other articles:
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J. Padilla, E. Daley, A. Chow, K. Robinson, K. Parthasarathi, A. N. J. McKenzie, T. Tschernig, V. P. Kurup, D. D. Donaldson, and G. Grunig IL-13 Regulates the Immune Response to Inhaled Antigens J. Immunol., June 15, 2005; 174(12): 8097 - 8105. [Abstract] [Full Text] [PDF]
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T. E. Toliver-Kinsky, W. Cui, E. D. Murphey, C. Lin, and E. R. Sherwood Enhancement of Dendritic Cell Production by Fms-Like Tyrosine Kinase-3 Ligand Increases the Resistance of Mice to a Burn Wound Infection J. Immunol., January 1, 2005; 174(1): 404 - 410. [Abstract] [Full Text] [PDF]
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K. A. Swanson, Y. Zheng, K. M. Heidler, Z.-D. Zhang, T. J. Webb, and D. S. Wilkes Flt3-Ligand, IL-4, GM-CSF, and Adherence-Mediated Isolation of Murine Lung Dendritic Cells: Assessment of Isolation Technique on Phenotype and Function J. Immunol., October 15, 2004; 173(8): 4875 - 4881. [Abstract] [Full Text] [PDF]
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J. A. Tobar, P. A. Gonzalez, and A. M. Kalergis Salmonella Escape from Antigen Presentation Can Be Overcome by Targeting Bacteria to Fc{gamma} Receptors on Dendritic Cells J. Immunol., September 15, 2004; 173(6): 4058 - 4065. [Abstract] [Full Text] [PDF]
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