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A Single Intratracheal Dose of the Growth Factor Fms-Like Tyrosine Kinase Receptor-3 Ligand Induces a Rapid Differential Increase of Dendritic Cells and Lymphocyte Subsets in Lung Tissue and Bronchoalveolar Lavage, Resulting in an Increased Local Antibody Production¹

Reinhard Pabst^2,*, Anke Lührmann^*, Ivo Steinmetz and Thomas Tschernig^*

Departments of ^* Functional and Applied Anatomy and Medical Microbiology, Medical School of Hannover, Hannover, Germany

	Abstract

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Repetitive doses of the growth factor Fms-like tyrosine kinase receptor-3 ligand (Flt3L) have resulted in increased numbers of dendritic cells (DC) in various organs, and the effect on protective or tolerogeneic responses in the gut wall has been documented in the literature. In this study, for the first time, Flt3L was locally applied in the trachea of rats using a single dose only. A dose-dependent increase not only of DC, but also of T lymphocytes (CD4⁺ and CD8⁺), was seen with a maximum on day 3. The effects on the cells in the lung interstitium and the bronchoalveolar space showed some differences. The use of tetanus toxoid as a model Ag applied intratracheally after the local Flt3L stimulation resulted in increased levels of specific IgA and IgG in the lung. Thus, this novel approach of locally stimulating APCs by topical application of a DC growth factor before applying the Ag offers a new vaccination strategy.

	Introduction

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No other cell of the immune system has attracted more interest in the last decade than the dendritic cell (DC),³ and there is growing evidence for a broad heterogeneity. DC are extremely powerful APCs that are not only found in lymphoid organs, e.g., lymph nodes (LN) and spleen, but also in many nonlymphoid organs such as the skin, gut wall, and respiratory tract (for review, see Ref. 1). DC have been classified based on their stage of maturation and anatomical compartments (2): 1) DC precursors in the bone marrow and blood; 2) recently immigrated, immature DC in nonlymphoid organs such as the tracheal epithelium; 3) maturing DC after Ag uptake and on their way, for example, in afferent lymphatics; and 4) mature, Ag-presenting DC in the draining LN. In respect to their effect on Th1 and Th2 lymphocytes, DC have also been divided into DC1 and DC2 or monocyte-derived and plasmacytoid DC, each characterized by a different set of surface markers (3).

The lung not only contains large numbers of DC, but many basic aspects of DC functions have also been studied in detail in the lung, for example, the short life span, the rapid increase in numbers after viral or bacterial infections, and the suppressive role of corticosteroids (see Ref. 4). Using fluorescent labeling, the rapid Ag uptake in the epithelium and the migratory route to the draining LN have been documented (5). In the LN, the DC can retain MHC class II/Ag peptide complexes for a prolonged period (6, 7). Also, large Ags, such as conidiae and hyphae of fungi, e.g. Aspergillus fumigatus, have been shown to be phagocytosed, cytokine production induced, and DC further matured, while they migrated to bronchial LN to induce specific Th cell immune reactions within the draining LN (8). Most in vivo experiments have been performed in rodents and data on human DC produced in vitro (9, 10) or the SCID mouse model (11). DC in the lung might be of relevance to induce protective immunity, but also tolerance to inhaled Ag by the IL-10 production of DC (12), depending on the stage of maturation of the DC, as recently proposed (13).

Thus, DC play a role in the modulation of immune responses, for example, in tumor immunology (14, 15), as well as in inducing adaptive immune reactions (16) and tolerance (17). It has been stressed that there is an urgent need to study DC functions in vivo, which is much easier now that growth factors such as GM-CSF, G-CSF, and in particular the Fms-like tyrosine kinase receptor-3 ligand (Flt3L) have been described to stimulate the production and mobilization of DC precursors in the bone marrow and blood (18, 19). Obviously, different growth factors stimulate precursors of different DC in vitro (20) or in vivo (21). Some experiments with Flt3L are of particular interest, as repetitive i.p. doses of this growth factor resulted in an expansion of DC in the gut wall. Thus, tolerance was achieved using very low doses of Ag (17). In another mouse model, the expanded numbers of DC in the gut wall after repeated doses of Flt3L resulted in an up-regulated active response to fed soluble Ag (16), indicating a potential application of Flt3L in designing an optimal mucosal immunization strategy.

Having recently described an expansion of DC, macrophages, and lymphocytes after a single intratracheal dose of a synthetic lipoprotein (macrophage-activating lipopeptide-2), which is physiologically produced by mycoplasms in the rat lung (22), and this topically applied compound also resulting in less lung metastasis in a tumor model (23), we wanted to test whether intratracheally applied Flt3L would have an effect on the number of DC, macrophages, and lymphocyte subsets not only in the lung interstitium, but also in the bronchial space, which is a clinically easily accessible compartment via the bronchoalveolar lavage (BAL). The functional relevance of the local immunostimulatory effect of Flt3L was tested by applying tetanus toxoid as a model Ag, and the IgA and IgG Ab titers were measured in the BAL and serum.

Not only a surprisingly rapid expansion of DC was found with different responses in the lung interstitium and BAL, but also an increase of lymphocyte subsets after this single local injection. Thus, Flt3L seems to affect DC precursor cells in the bone marrow and to induce the expansion and probably also the maturation locally in the lung with an adjuvant effect on local Ab production.

	Materials and Methods

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Animals

Adult (6–8 wk of age) Lewis rats were obtained from the central animal laboratory of the Medical School of Hannover and were kept under specific pathogen-free conditions. The animals had free access to pelleted food and water and were housed under standardized light/dark cycle. The number of animals per group can be seen in the figures (mean weight dose response: males 217 ± 5.2 g (SEM), time sequence 284 ± 13 g, i.p. dose 228 ± 6.5 g, tetanus toxoid immunization 182 ± 2 g, female animals).

Intratracheal instillation

Under short ether anesthesia, rats were suspended in a hanging position by a rubber band fixed to the incisor teeth of the upper jaw. The trachea was intubated via the oral cavity, a tube (17G; Braun, Melsungen, Germany) was placed in the trachea, the drug or saline was instilled, and 500 µl air was blown into the lung.

Stimulation by Flt3L

Human rFlt3L provided by Immunex (Seattle, WA) was instilled intratracheally at a dose of 1, 10, or 100 µg per animal or i.p. (100 µg).

Immune response to tetanus toxoid

Tetanus toxoid (Tetanol; Chiron Behring, Marburg, Germany) was instilled intratracheally (20 intraunits, corresponding to 250 µl). The animals were instilled with saline (as a control) or 100 µm Flt3L in a total volume of 250 µl of saline on day 0, received tetanus toxoid on days 3 and 7, and were sacrificed on day 14. The content of IgA and IgG Abs was determined in 5 ml of BAL fluid and serum samples in parallel.

ELISA methods

Single U-shaped wells of nonflexible polystyrol microtiter plates were coated for 2 h with 10 µl tetanus toxoid (3 µg protein/ml; Calbiochem-Novabiochem, Bad Soden, Germany) diluted in 10 mM PBS, pH 7.4. After a washing step with PBS and a blocking step with PBS-BSA (PBS containing 1% (w/v) BSA) for 30 min, rat sera were diluted in PBS-BSA and 20 µl was incubated for 2 h. For the determination of the different isotypes, plates were washed and incubated with 10 µl of either biotin-labeled goat anti-rat IgG (1/1000 in PBS-BSA; Southern Biotechnology Associates, Birmingham, AL) or goat anti-rat IgA (1/1000; Serotec, Oxford, U.K.) for 60 min. Microtiter plates were then developed with streptavidin coupled to -galactosidase. The 4-methylumbelliferyl--D-galactopyranoside was used as substrate, and the fluorescent product was measured in a Titertek Fluoroscan 2 (Huntsville, AL; excitation 355 nm, emission 460 nm) as relative fluorescence units. For IgG, a high titered anti-tetanus toxoid IgG rat serum was used as a standard. For each assay, a standard curve was generated using appropriate dilutions of this serum. The relative fluorescence unit value of the highest IgG Ab concentration falling within the linear portion of the different standard curves was arbitrarily set as 10 ELISA units. The specific IgG concentration of each serum sample was determined from the standard regression curve constructed for each assay and expressed as ELISA units.

Preparation of cells of the bronchoalveolar space

BAL was performed by instilling 10 x 5 ml of cold saline via a tracheal cannula. The recovered lavage fluid (>90% of the instilled volume) was pooled and centrifuged at 400 x g for 10 min. Cells were washed and resuspended in 1 ml PBS containing 1% BSA and 0.1% sodium azide. The number of nucleated cells was determined by a Neubauer counting chamber. Differential counts were done on cytospots (fixed in acetone for 10 min and washed with TBS containing 0.1% Tween 20).

Preparation of cells of the lung interstitium

The left lungs were dissected and then passed through a metal sieve with two rounded tweezers. After rinsing the sieve with 40 ml of PBS, the cell suspensions were centrifuged at 400 x g for 10 min. The interstitial cells were processed, as described for the BAL cells.

Staining of leukocyte subsets and DC

For flow cytometry, cells were transferred into microtiter plates (10⁶ cells/well) and washed twice in PBS containing 1% BSA and 0.1% NaN₃. After incubation with the first mAbs for 30 min at 4°C, the cells were washed twice and the secondary Abs were incubated for the same time and temperature. The cells were analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA), focusing on the lymphocyte cluster and the DC. CD8⁺ T cells were identified as R73⁺, Ox8⁺; CD4⁺ T cells as R73⁺, Ox8^-; activated CD4 cells as R73⁺, Ox8^-, and Ox39⁺. Isotype-matched Abs served as a control. DC were identified as low-autofluorescence cells, non-T and non-B lymphocytes, and double positive for Ox6 (MHC class II) and Ox62 (DC marker) (24). For characterization of the MHC class II/Ox62⁺ DC, cells were stained with Abs against CD80, CD86, CD11c, and CD54, and the mean fluorescence intensity (MFI) was measured. MFI data were corrected for appropriate isotype controls IgG1 and IgG2a. All primary Abs were monoclonal mouse anti-rat (Serotec). Unconjugated Abs were detected using PE-conjugated secondary Ab (PE; Dianova, Hamburg, Germany) and biotinylated primary Abs with Red 670-streptavidin (Life Technologies, Gaithersburg, MD). The neutrophils were morphologically identified by May-Grünwald/Giemsa staining.

Histology

Samples of the right lung were fixed in 4% buffered Formalin and embedded in paraffin, and the sections (5 µm) were stained with H&E.

Immunohistology

Cryosections of lung tissue (6 µm thick) were made. After air drying, slides were fixed in acetone for 10 min, washed in TBS containing 0.05% Tween 20 (Serva, Heidelberg, Germany), and incubated for 30 min in a moist chamber with the primary Abs (Ox6 and Ox62). They were then incubated with a second Ab (rabbit anti-mouse Ig, Z259; DAKO, Hamburg, Germany) and an Ab complex (alkaline phosphatase-antialkaline phosphatase complex, D651; DAKO) for 30 min. After repeating the last two steps for 15 min, 2 mg fast blue (Sigma-Aldrich, Munich, Germany), mixed with 4 ml alkaline phosphatase-antialkaline phosphatase substrate (9.8 ml 0.1 Tris buffer (pH 8.2) containing 2 mg naphthol AS-MX phosphate, 200 ml N,N-dimethylformamide, and 10 µl 1 M levamisole), was added to the slides for 25 min. Slides were washed and counterstained with hematoxylin (Fluka, Buchs, Switzerland) and mounted in glycergel (DAKO).

Data analysis

Means, SEs, and the level of significance were determined taking ≤0.05 as statistically significant using SPSS-Windows (SPSS, Chicago, IL) and applying the Mann-Whitney U test.

	Results

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A single dose of 100 µg Flt3L resulted in an enlargement of the peribronchovascular space and a moderate leukocyte infiltration around small bronchi and branches of the pulmonary arteries, with a maximum on day 3 (Fig. 1). Using immunohistology, the increase of DC in the wall of bronchi could be seen after Flt3L (Fig. 1D). With Abs against MHC class II and Ox62, a double staining was documented for these cells (Fig. 1, G and H).

View larger version (137K): [in this window] [in a new window]   FIGURE 1. Histological sections of a rat lung 3 days after intratracheal saline injection (A), after Flt3L (100 µg) injection 3 days (B) or 10 days before (C). Immunohistology of a rat lung 3 days after saline (D) or Flt3L (F) instillation, MHC class II staining. A control section stained without the primary Ab is documented in E. There are more stained cells after Flt3L. On sequential sections, Ox62 was used to show DC (G), and a double staining of Ox62 and MHC class II is documented in H. The bar represents 100 µm (A–C) and 50 µm (D–H). The arrows indicate bronchi, and the arrowheads accompanying pulmonary arteries.

There were no significant differences between animals injected with 1 and 10 µg Flt3L when the total number of leukocytes, lymphocytes, or DC was compared. However, the dose of 100 µg intratracheally resulted in significantly more leukocytes, lymphocytes, and DC in the lung interstitium, but in the BAL only DC were significantly increased after a dose of 10 µg (Fig. 2). When the DC from the lung interstitium were separated and the MHC class II/Ox62⁺ cells further characterized, other DC markers such as CD80, CD86, CD11c, and CD54 could be documented on different subsets (Fig. 3). The number of neutrophils did not show an increase at any time after local Flt3L instillation. Rats receiving the same single dose of 100 µg systemically by an i.p. injection showed no effect on the total numbers. The relative number of leukocyte subsets did not differ (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Total numbers of leukocytes, lymphocytes, and DC in the lung interstitial tissue and the BAL on day 3 after different doses of Flt3L. The asterisk indicates significant differences.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Cells of the lung tissue were separated and investigated by flow cytometry (n = 5). The MFI of CD80, CD86, CD11c, and CD54 expression of double-positive cells for MHC class II and Ox62 DC markers was determined after correction of the isotype control.

In the interstitial space of the lung in contrast to the BAL, a significant increase of the total leukocyte number was seen on day 3 after Flt3L instillation (Fig. 4): this increase was not only caused by more DC, but also by lymphocytes and the CD4 and CD8 subsets (Fig. 5). The kinetics of the increase of the different leukocyte subsets differed between the interstitial compartment and the BAL, in which the maximum was seen on day 3, followed by a drop to day 10.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Comparison of the total number of leukocytes in the lung tissue and BAL at different times after 100 µg Flt3L intratracheal (i.t.) instillation, saline instillation, or application of the same dose of Flt3L i.p. The asterisk indicates significant differences (x ± SEM).

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Lymphocyte and DC subsets at different times after the application of 100 µg Flt3L either i.p. or intratracheally (i.t.). In a control group, saline was instilled into the trachea. The asterisk indicates significant differences. # = no cells (x ± SEM).

Previous Flt3L stimulation resulted in significantly higher IgA antitoxoid concentrations in the BAL compared with controls (Fig. 6). No differences were seen in specific serum IgA. The IgG anti-tetanus toxoid concentrations were also significantly increased after Flt3L stimulation in the BAL fluid. However, in contrast to IgA, there was also a significant increase of specific IgG in the serum, although less pronounced compared with the BAL (Fig. 6).

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Specific anti-tetanus toxoid IgA and IgG levels in the BAL and the sera of animals after intratracheal immunization with tetanus toxoid. Animals were treated intratracheally with either Flt3L or saline before immunization. The asterisk indicates significant differences.

	Discussion

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To date, Flt3L has only been tested in vitro or after repetitive doses were given systemically (e.g., Refs. 16, 17). In this study, we show for the first time that a local application resulted in a significant increase of DC, which had a stimulatory effect on local Ab production. Most experimental studies on the effects of Flt3L have been performed in the mouse, and thus the dose applied in rats was extrapolated on a weight basis. It is not known where in the lung the Flt3L is absorbed and how it reaches the interstitial space to influence the DC in this compartment. In future studies, whole mount preparations will be used, as described for the mouse lung, to study the DC in the tracheal and bronchial epithelial layer. The MHC class II/Ox62⁺ DC expressed other markers for DC of differential maturation stages to a varying extent: few cells were also positive for CD80 and CD86, while others expressed a high density of CD11c and CD54 (Fig. 3) with a pattern comparable to that shown for rat DC previously (26, 27).

The increase in the number of DC after the local Flt3L instillation into the trachea may be caused by local proliferation, maturation, or reduced emigration into the draining LN. The bone marrow as a source of these DC, as is likely after repetitive doses systematically applied (17), can be excluded because the same single dose applied i.p. did not result in any effects in the lung.

The differential results regarding the number of leukocytes and their subsets in the lung interstitium and the bronchoalveolar space support our concept that the BAL should not be automatically considered as representing the situation in the lung as a whole (28). After Flt3L treatment, the DC have to be characterized further by using other markers, because the evidence is continuously growing that DC are extremely heterogeneous in surface Ag expression and functional aspects.

The increase of lymphocytes after a local Flt3L instillation was surprising, as to date reports of the effects have focused on DC (18, 19, 21). The lymphocyte subset composition differs between the various compartments of the lung, and little is known about regulatory factors, e.g., adhesion molecules, chemokines, or their receptors, to direct the lymphocytes from one compartment of the lung to the other. However, cytokine production by lymphocytes, e.g., IFN- (for review, see Ref. 29), differs between lung compartments in immune reactions (30). It has to be stressed that only in the lung do lymphocytes traverse a body surface, return later to the organ, and migrate finally to the draining, i.e., the bronchial LN (31). Recently, we proposed a concept of a unique compartment in the lung, the perivascular space (32). It was surprising to find an enlargement of this space after the intratracheal Flt3L application, with a maximum on day 3 (Fig. 1). The functional consequences of this phenomenon have to be studied in future. Flt3L might stimulate certain cells in the lung, which then secrete cytokines to modify the local microenvironment to attract other leukocyte subsets. If the increase of lymphocytes were caused by factors produced by DC, one would expect different time kinetics with an initial increase of DC, followed by the increase of lymphocytes. That was not the case (Fig. 4). Obviously, the local regulation is more complex. By serial analysis of gene expression of gut intraepithelial TCR⁺ and TCR⁺ lymphocytes, a moderately increased expression of Flt3L was documented (33). Similar mechanisms might also play a role in the lung.

The increased local Ab production in the lung after local Flt3L stimulation may be of great clinical relevance. IgA as the typical Ab of mucosal organs (34) will prevent the adhesion of bacteria to the epithelium of the trachea and bronchi. IgA is produced by plasma cells in the lamina propria and transported to the bronchial lumen by the poly Ig receptor. In the peripheral parts of the lung, IgG is of great relevance. IgG produced by local plasma cells in the lamina propria and probably also in the draining bronchial LN reaches the serum via the lymphatics and can also diffuse into the air space, from where it was recovered by the BAL.

The previous experiments with repetitive systemic doses of Flt3L resulted in an increased production and emigration of DC to all lymphoid and nonlymphoid organs, which can be advantageous for certain diseases. However, in most situations, it would be preferable to induce an immune response (either protective and tolerogenic) at a specific site in which an Ag as a vaccine would be applied, and not affecting all Ags that gain access to the immune system at that time, e.g., microbial Ags of the gut and other mucosal sites. Future studies have to clarify whether repetitive doses of Flt3L or other growth factors, such as GM-CSF, would be even more effective in inducing tolerogenic or immunogenic protective responses. After it became obvious that the DC subset composition in the blood of children differs with age (35) and how important the local stimulation of the lung by environmental factors is, e.g., LPS in early childhood for the development of asthma, one might speculate on prophylactic stimulation of the lung by inhaling stimulatory factors for lung DC using compounds such as Flt3L. A further application might be to induce antitumor activity by the combination of Flt3L and immunostimulatory DNA or a tumor Ag, as suggested by Merad et al. (36) recently.

	Acknowledgments

 
We thank Immunex for the Flt3L, Birgit Brenneke, Britta Wenske, and Karin Westermann for excellent technical assistance, and Dr. C. Kruschinski for help with the double staining. The correction of the English text by Sheila Fryk is gratefully acknowledged.

	Footnotes

 
¹ This work was supported by grants from the German Research Foundation (DFG Pa 240/8-2 and SFB 587, B1).

² Address correspondence and reprint requests to Dr. Reinhard Pabst, Center of Anatomy 4120, Medical School of Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail address: pabst.reinhard{at}mh-hannover.de

³ Abbreviations used in this paper: DC, dendritic cell; BAL, bronchoalveolar lavage; Flt3L, Fms-like tyrosine kinase receptor-3 ligand; LN, lymph node; MFI, mean fluorescence intensity.

Received for publication November 7, 2002. Accepted for publication April 29, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Lipscomb, M. F., B. J. Masten. 2002. Dendritic cells: immune regulators in health and disease. Physiol. Rev. 82:97.[Abstract/Free Full Text]
2. Austyn, J. M.. 1996. New insights into the mobilization and phagocytic activity of dendritic cells. J. Exp. Med. 183:1287.[Free Full Text]
3. Rissoan, M. C., V. Soumelis, N. Kadowaki, G. Grouard, F. Briere, R. de Waal-Malefyt, Y. J. Liu. 1999. Reciprocal control of T helper cell and dendritic cell differentiation. Science 283:1183.[Abstract/Free Full Text]
4. Holt, P. G., P. A. Stumbles. 2000. Regulation of immunologic homeostasis in peripheral tissues by dendritic cells: the respiratory tract as a paradigm. J. Allergy Clin. Immunol. 105:421.[Medline]
5. Vermaelen, K. Y., I. Carro-Muino, B. N. Lambrecht, R. A. Pauwels. 2001. Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes. J. Exp. Med. 193:51.
6. Vu, Q., K. M. McCarthy, J. M. McCormack, E. E. Schneeberger. 2002. Lung dendritic cells are primed by inhaled particulate antigens, and retain MHC class II/antigenic peptide complexes in hilar lymph nodes for a prolonged period of time. Immunology 105:488.[Medline]
7. Julia, V., E. M. Hessel, L. Malherbe, N. Glaichenhaus, A. O’Garra, R. L. Coffman. 2002. A restricted subset of dendritic cells captures airborne antigens and remains able to activate specific T cells long after antigen exposure. Immunity 16:271.[Medline]
8. Bozza, S., R. Gaziano, A. Spreca, A. Bacci, C. Montagnoli, P. di Francesco, L. Romani. 2002. Dendritic cells transport conidia and hyphae of Aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate Th responses to the fungus. J. Immunol. 168:1362.[Abstract/Free Full Text]
9. Cochand, L., P. Isler, F. Songeon, L. P. Nicod. 1999. Human lung dendritic cells have an immature phenotype with efficient mannose receptors. Am. J. Respir. Cell Mol. Biol. 21:547.[Abstract/Free Full Text]
10. De Yang, Q. C., O. Chertov, J. J. Oppenheim. 2000. Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J. Leukocyte Biol. 68:9.[Abstract/Free Full Text]
11. Hammad, H., C. Duez, O. Fahy, A. Tsicopoulos, C. André, B. Wallaert, S. Lebecque, A. B. Tonnel, J. Pestel. 2000. Human dendritic cells in the severe combined immunodeficiency mouse model: their potentiating role in the allergic reaction. Lab. Invest. 80:605.[Medline]
12. Akbari, O., R. H. DeKruyff, D. T. Umetsu. 2001. Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat. Immun. 2:725.[Medline]
13. Lutz, M. B., G. Schuler. 2002. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?. Trends Immunol. 23:445.[Medline]
14. Fong, L., Y. Hou, A. Rivas, C. Benike, A. Yuen, G. A. Fisher, M. M. Davis, E. G. Engleman. 2001. Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy. Proc. Natl. Acad. Sci. USA 98:8809.[Abstract/Free Full Text]
15. Averbook, B. J., J. L. Schuh, R. Papay, C. Maliszewski. 2002. Antitumor effects of Flt3 ligand in transplanted murine tumor models. J. Immunother. 25:27.
16. Williamson, E., G. M. Westrich, J. L. Viney. 1999. Modulating dendritic cells to optimize mucosal immunization protocols. J. Immunol. 163:3668.[Abstract/Free Full Text]
17. Viney, J. L., A. M. Mowat, J. M. O’Malley, E. Williamson, N. A. Fanger. 1998. Expanding dendritic cells in vivo enhances the induction of oral tolerance. J. Immunol. 160:5815.[Abstract/Free Full Text]
18. Pulendran, B., E. Maraskovsky, J. Banchereau, C. Maliszewski. 2001. Modulating the immune response with dendritic cells and their growth factors. Trends Immunol. 22:41.[Medline]
19. McKenna, H. J.. 2001. Role of hematopoietic growth factors/flt3 ligand in expansion and regulation of dendritic cells. Curr. Opin. Hematol. 8:149.[Medline]
20. Gilliet, M., A. Boonstr, C. Paturel, S. Antonenko, X. L. Xu, G. Trinchieri, A. O’Garra, Y. J. Liu. 2002. The development of murine plasmacytoid dendritic cell precursors is differentially regulated by Flt3-ligand and granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 195:953.[Abstract/Free Full Text]
21. Maraskovsky, E., K. Brasel, M. Teepe, E. R. Roux, S. D. Lyman, K. Shortman, H. J. McKenna. 1996. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J. Exp. Med. 184:1953.[Abstract/Free Full Text]
22. Lührmann, A., U. Deiters, J. Skokowa, M. Hanke, J. E. Gessner, P. F. Mühlradt, R. Pabst, T. Tschernig. 2002. In vivo effects of a synthetic 2-kilodalton macrophage-activating lipopeptide of Mycoplasma fermentans after pulmonary application. Infect. Immun. 70:3785.[Abstract/Free Full Text]
23. Shingu, K., C. Kruschinski, A. Lührmann, K. Grote, T. Tschernig, S. von Hörsten, R. Pabst. 2003. Intratracheal macrophage-activating lipopeptide-2 (MALP-2) reduces metastasis in the rat lung. Am. J. Respir. Cell Mol. Biol. 28:316.[Abstract/Free Full Text]
24. Lambrecht, B. N., I. Carro-Muino, K. Vermaelen, R. A. Pauwels. 1999. Allergen-induced changes in bone-marrow progenitor and airway dendritic cells in sensitized rats. Am. J. Respir. Cell Mol. Biol. 20:1165.[Abstract/Free Full Text]
25. Parajuli, P., R. L. Mosley, V. Pisarev, J. Chavez, A. Ulrich, M. Varney, R. K. Singh, J. E. Talmadge. 2001. Flt3 ligand and granulocyte-macrophage colony-stimulating factor preferentially expand and stimulate different dendritic and T-cell subsets. Exp. Hematol. 29:1185.[Medline]
26. Stumbles, P. A., J. A. Thomas, C. L. Pimm, P. T. Lee, T. J. Venaille, S. Proksch, P. G. Holt. 1998. Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2 (Th2) responses and require obligatory cytokine signals for induction of Th1 immunity. J. Exp. Med. 188:2019.[Abstract/Free Full Text]
27. Stumbles, P. A., D. H. Strickland, C. L. Pimm, S. F. Proksch, A. M. Marsh, A. S. McWilliam, A. Bosco, I. Tobagus, J. A. Thomas, S. Napoli, et al 2001. Regulation of dendritic cell recruitment into resting and inflamed airway epithelium: use of alternative chemokine receptors as a function of inducing stimulus. J. Immunol. 167:228.[Abstract/Free Full Text]
28. Pabst, R., T. Tschernig. 1997. Lymphocyte dynamics: caution in interpreting BAL numbers. Thorax 52:1078.[Abstract]
29. Pabst, R.. 2000. Mucosa-associated lymphoid tissue of the lung: localization, numbers and dynamics of lymphoid cells in the five different compartments. W. Busse, and S. T. Holgate, eds. Asthma and Rhinitis 543. Blackwell, Oxford.
30. Klemm, A., T. Tschernig, N. Krug, R. Pabst. 1998. Lymphocyte subsets in distinct lung compartments show a different ability to produce interferon- (IFN) during a pulmonary immune response. Clin. Exp. Immunol. 113:252.[Medline]
31. Lehmann, C., A. Wilkening, D. Leiber, A. Markus, N. Krug, R. Pabst, T. Tschernig. 2001. Lymphocytes in the bronchoalveolar space reenter the lung tissue by means of the alveolar epithelium, migrate to regional lymph nodes, and subsequently rejoin the systemic immune system. Anat. Rec. 264:229.[Medline]
32. Pabst, R., T. Tschernig. 2002. Perivascular capillaries in the lung: an important but neglected vascular bed in immune reactions?. J. Allergy Clin. Immunol. 110:209.[Medline]
33. Shires, J., E. Theodoridis, A. C. Hayday. 2001. Biological insights into TCR⁺ and TCR⁺ intraepithelial lymphocytes provided by serial analysis of gene expression (SAGE). Immunity 15:419.[Medline]
34. Russell, M. W., M. Kilian, M. E. Lamm. 1999. Biological activities of IgA. P. L. Ogra, and J. Mestecky, and M. E. Lamm, and W. Strober, and J. Bienenstock, and J. R. McGhee, eds. Mucosal Immunology 225. Academic Press, San Diego.
35. Teig, N., D. Moses, S. Gieseler, U. Schauer. 2002. Age-related changes in human blood dendritic cell subpopulations. Scand. J. Immunol. 55:453.[Medline]
36. Merad, M., T. Sugie, E. G. Engleman, L. Fong. 2002. In vivo manipulation of dendritic cells to induce therapeutic immunity. Blood 99:1676.[Abstract/Free Full Text]

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