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Lung Dendritic Cells Elicited by Fms-like Tyrosin 3-Kinase Ligand Amplify the Lung Inflammatory Response to Lipopolysaccharide

¹ Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Giessen Lung Center, Giessen, Germany; and ² Department of Pulmonary Medicine, Hannover School of Medicine, Hannover, Germany

Correspondence and requests for reprints should be addressed to Ulrich A. Maus, Ph.D., Hannover School of Medicine, Department of Pulmonary Medicine, Laboratory for Experimental Lung Research, Hannover 30625, Germany. E-mail: maus.ulrich{at}mh-hannover.de

	ABSTRACT

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Rationale: Strategically located beneath the alveolar epithelial barrier, dendritic cells (DCs) of the lung are centrally involved in the sampling and processing of inhaled antigens. However, the contribution of DCs to acute lung inflammation induced by inhaled bacterial toxins is largely unknown.

Objectives: To determine the effect of increased lung DC numbers elicited by Fms-like tyrosine kinase-3 ligand (Flt3L) on the acute lung inflammatory response to Escherichia coli lipopolysaccharide (LPS) and Klebsiella pneumoniae infection.

Methods: Mice were pretreated with Flt3L either in the absence or presence of anti-CD11a antibodies to block the Flt3L-elicited lung DC accumulation or were made transiently neutropenic and then challenged with E. coli LPS or K. pneumoniae.

Measurements and Main Results: Flt3L-pretreated mice challenged with LPS responded with drastically increased numbers of both lung parenchymal and alveolar DCs together with an aggravated neutrophilic alveolitis, elevated tumor necrosis factor- and IL-12 levels, and a strongly increased lung permeability compared with LPS- or Flt3L-only–treated mice. Anti–CD11a-mediated blockade of lung DC accumulation significantly attenuated the lung permeability developing in response to LPS, whereas transient neutropenia did not affect lung permeability changes. Finally, Flt3L-pretreated mice responded with increased lung permeability and decreased survival upon infection with K. pneumoniae.

Conclusions: Lung DCs actively participate in the early inflammatory response to both inhaled bacterial toxins and live bacteria and play a yet unrecognized role in regulating lung barrier integrity.

Key Words: dendritic cell • lung • inflammation • neutrophil • monocyte

AT A GLANCE COMMENTARY TOP ABSTRACT AT A GLANCE COMMENTARY METHODS RESULTS DISCUSSION REFERENCES   Scientific Knowledge on the Subject Little is known about the contribution of dendritic cells to the lung inflammatory response to inhaled bacterial toxins. What This Study Adds to the Field Lung dendritic cells have a role in the regulation of lung barrier integrity that is independent of the lung neutrophil response in a murine model of endotoxin-induced inflammation.

 
Dendritic cells (DCs) of the lung are ideally situated in close proximity to alveolar epithelium and resident alveolar macrophages to sample and process inhaled antigens, thus rendering these cells critical to the initiation or suppression of adaptive immune responses of the lung (1–3). In addition to acting as antigen-presenting cells, lung DCs have been shown to express a multitude of Toll-like receptors (TLRs) (4), allowing them to respond to TLR ligands with the release of proinflammatory cytokines such as tumor necrosis factor (TNF)- and IL-12, thereby promoting proinflammatory responses. On the other hand, DCs have also been reported to suppress inflammatory responses by their release of immunoregulatory cytokines such as IL-10 and transforming growth factor (TGF)- (1, 5). Although it is generally assumed that resident alveolar macrophages are central to the initiation and propagation of lung inflammation developing in response to bacterial toxins and infections, the in vivo contribution of lung DCs under these conditions has so far not been examined. This is probably due to the low numbers of DCs found in the lung parenchymal tissue under steady-state conditions. However, recent reports have demonstrated that systemic administration of Fms-like tyrosine 3-kinase ligand (Flt3L), a hematopoietic growth factor involved in the differentiation of DCs from hematopoietic stem cells, leads to a drastic increase in functionally active DCs in a variety of organs, including the lung (6–9). As opposed to the beneficial effects of Flt3L-elicited DCs on airway inflammation, such as asthma, and systemic infections with intracellular pathogens, such as Listeria monocytogenes (5, 10, 11), little is known about the lung DC pathobiology in mice in response to treatment with bacterial toxins. In the current study, we set out to define the contribution of DCs to lung inflammation developing in response to Escherichia coli endotoxin and Klebsiella pneumoniae challenge. Systemic application of Flt3L led to a 2 integrin–dependent accumulation of DCs in the lung parenchyma but not in the alveolar airspace of mice without inducing lung inflammation. However, Flt3L-pretreated mice responded with a severely aggravated lung inflammation upon E. coli LPS challenge and K. pneumoniae infection, and this was attributable to the Flt3L-elicited DCs rather than the neutrophilic component of lung inflammation. These data demonstrate that lung DCs may be a critical component of lung inflammation elicited by bacterial toxins or live bacteria and show a previously unrecognized role of these cells in the regulation of lung barrier integrity. Some of the results of this study have been previously reported as an abstract (12).

	METHODS

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Mice
Wild-type C57BL/6 mice (18–22 g) were purchased from Charles River (Sulzfeld, Germany) and kept under conventional conditions with free access to food and water. All animal experiments were approved in accordance with the guidelines of our local government authority.

Reagents, Flow Cytometry, and Cell Sorting
The panel of anti-mouse monoclonal antibodies (mAbs) used for flow cytometric analyses, the function-blocking antibodies used, and the protocol to deplete circulating neutrophils are outlined in the online supplement. Flt3L was a kind gift from Amgen, Inc. Ultrapure E. coli O111:B4 lipopolysaccharide (LPS) was purchased from Calbiochem (La Jolla, CA). Staining of cells and flow cytometry using a FACSCanto and a FACSVantage SE flow cytometer (BD Biosciences, San Jose, CA) are outlined in detail in the online supplement.

Treatment Protocols and Analysis of the Inflammatory Phenotype
Mice were treated systemically with Flt3L (10µg in 100 µl phosphate-buffered saline [PBS]/0.1% human serum albumin) or vehicle daily for the indicated time points in the absence or presence of function-blocking antibodies or isotype controls. Subsequently, lung inflammation was induced by intratracheal application of LPS (1 µg ultrapure LPS/mouse), as described (13). In selected experiments, circulating neutrophils were depleted using anti–Gr-1 mAbs (14). For induction of peritoneal inflammation, mice were injected intraperitoneally with 20 µg LPS in a total volume of 100 µl sterile normal saline. For infection experiments, K. pneumoniae (American Type Cell Culture no. 43861) was propagated as described in the online supplement. On Day 9 of Flt3L or vehicle treatment, mice received an intratracheal application of 10⁴ cfu K. pneumoniae suspended in 70 µl sterile PBS. Lung leakage was analyzed by intravenous application of fluorescein isothiocyanate (FITC) albumin (Sigma, Taufkirchen, Germany) (15). Analysis of the inflammatory phenotype was performed as described previously (13, 15) (see online supplement).

Isolation and Identification of Lung and Alveolar DCs
Isolation and identification of lung DCs were performed as described in detail previously (16, 17), with some modifications. Briefly, lavaged and perfused lungs were digested and homogenized. CD11c-positive cells were purified from the resultant lung homogenates using magnetic beads and analyzed by flow cytometry. In some experiments, DCs from lung homogenates were flow-sorted for a subsequent in vitro stimulation of sorted DCs with LPS or for gene expression analysis of proinflammatory cytokines subsequent to LPS application in vivo (see online supplement).

RNA Isolation and Polymerase Chain Reaction
RNA isolation from sorted DCs, cDNA synthesis, reverse transcriptase–polymerase chain reaction (RT-PCR), and real-time RT-PCR analyses were performed as outlined in the online supplement. PBGD (porphobilinogen deaminase), or HMBS (hydroxymethyl-bilane synthase) and -actin served as the reference genes in real-time RT-PCR runs. Fold-changes in gene expression analyses were determined using the 2^–Ct method, as recently described (18).

Immunohistochemistry
Perfused lungs were inflated with TissueTek OCT (Sakura Finetek, Zoeterwoude, The Netherlands) and snap-frozen in liquid nitrogen. Lung tissue cryosections (7 µm) were stained for CD11c, CD11b, and F4/80 expression using alkaline phosphatase and Fast-Red substrate (DakoCytomation, Hamburg, Germany), and counterstained with hemalaun (see online supplement).

ELISA and Protein Concentration Measurement
Cytokine concentrations in bronchoalveolar lavage fluid (BALF) were determined using DuoSet ELISA plates (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. Total protein concentration in BALF was measured using the Bradford reagent kit (BioRad, Hercules, CA).

Statistics
Data analysis and statistics were performed using SPSS version 12.0 (SPSS, Inc., Chicago, IL). All data are displayed as mean values ± SD. Statistical differences between treatment groups were estimated by Kruskal-Wallis test, followed by Mann-Whitney U test. Differences were considered statistically significant when P values were less than 0.05.

	RESULTS

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Identification and Characterization of Lung DCs
In normal mouse lung parenchymal tissue, two major populations of CD11c-positive cells have been reported, including lung DCs and lung macrophages (2, 17). Among the lung DCs, two subpopulations have been identified, including myeloid DCs and plasmacytoid DCs, of which the myeloid DC subset is by far the predominating DC population (>95%) (17). Myeloid DCs are characterized by their low autofluorescence and CD11b⁺, CD11c⁺, MHCII⁺, CD86⁺, and CD205^– cell surface antigen expression profile. In contrast, plasmacytoid DCs are CD11c⁺ and B220⁺, but CD11b^–. As opposed to lung DCs, CD11c-positive lung macrophages are highly autofluorescent, and are MHCII^+/–, CD11b^–, and CD86^–. In the current study, CD11c-positive cells with low autofluorescent properties purified from lung homogenates of untreated mice were identified to be MHCII⁺, CD205^–, CD11b⁺, and CD86⁺, thus representing myeloid DCs. However, in response to Flt3L treatment, in addition to myeloid DCs, a small but detectable number of plasmacytoid DCs (CD11c⁺, CD11b^–, CD8^–, B220⁺) and lymphoid DCs (CD11c⁺, CD11b^–, CD8⁺, B220^–) were detected in lung homogenates (Figure 1B). Fluorescence-activated cell sorter (FACS) analysis of the highly autofluorescent CD11c-positive cells collected from lung homogenates of untreated and Flt3L-treated mice revealed a CD11c⁺, MHCII^–, CD205^–, CD11b^–, CD86^–, F4/80⁺ immunophenotype, thus representing "classical" lung macrophages (Figures 1A and 1B). Collectively, and in agreement with previous reports, CD11c-positive cells collected from lung homogenates of untreated C57BL/6 mice largely consist of lung myeloid DCs and lung macrophages, whereas Flt3L treatment of mice primarily results in an expansion of lung myeloid DCs accompanied by a low but detectable increase in numbers of lymphoid and plasmacytoid DCs.

View larger version (75K): [in this window] [in a new window]   Figure 1. Identification and characterization of lung parenchymal dendritic cells (DC) and lung macrophages. Mice were treated systemically for 9 consecutive days with Flt3L (10 µg/mouse/d) or vehicle. Subsequently, CD11c-positive cells in lung homogenates were purified by magnetic cell sorting and analyzed by flow cytometry. (A) Fluorescence-activated cell sorter (FACS) analysis of purified CD11c-positive cells from lung homogenates of vehicle-treated mice (left dot plots) and Flt3L-pretreated mice (right dot plots) discriminated lung DCs from lung macrophages according to their differences in scatter characteristics and autofluorescence (FL1) properties. (B) FACS analysis of cell surface antigen distribution profiles of lung DCs and lung macrophages collected from lung parenchymal tissue of vehicle-treated mice (left histograms in B) and Flt3L-pretreated mice (right histograms in B). The histograms in B represent the indicated cell surface antigen expression profiles of lung DCs and lung macrophages gated according to their differential scatter and fluorescence 1 characteristics as shown in A (open histograms, controls; shaded histogram overlays, specific antigen expression, as indicated). The data are representative of five independent experiments. FSC = forward scatter; lung M = lung macrophages; SSC = side scatter.

View larger version (25K): [in this window] [in a new window]   Figure 2. Time course of Flt3L-elicited lung dendritic cell (DC) accumulation. Mice were either left untreated or received a daily injection of Flt3L for the indicated time intervals. Subsequently, mice were killed and CD11c-positive lung DCs and lung macrophages were collected from the lung parenchymal tissue. (A) Proportions of CD11c-positive lung DCs and lung macrophages as differentiated according to their autofluorescent properties. The cells with low autofluorescent properties represent lung DCs and cells with high autofluorescent properties represent lung macrophages. (B) Total numbers of lung DCs and lung macrophages (M) contained in lung homogenates of vehicle-treated or Flt3L-treated mice, as indicated. Values in B show mean ± SD of n = 3 to 5 animals per time point. *Signifies P < 0.05 compared with control.

View larger version (32K): [in this window] [in a new window]   Figure 3. Analysis of the molecular pathways of Flt3L-induced lung dendritic cell (DC) accumulation. Mice were treated with Flt3L or vehicle for 9 days (10 µg/mouse/d) and concomitantly received function-blocking monoclonal antibodies with specificity for the indicated cell adhesion molecules, or isotype control IgG. (A) Total numbers of lung DCs recovered from lung homogenates of untreated versus Flt3L-treated mice were calculated as outlined in Figure 2. (B) Percentages of DCs of total cells from mediastinal lymph nodes and spleens. Note that, in contrast to lung DC recruitment, DC percentages in mediastinal lymph nodes or in spleens were not affected by antibody treatment. Values are presented as mean ± SD of n = 3 to 5 animals per treatment group. $Signifies P < 0.05 compared with Flt3L-treated isotype control; *Signifies P < 0.05 compared with vehicle-treated mice. Isotp = isotype control IgG.

View larger version (66K): [in this window] [in a new window]   Figure 4. Effect of 2 integrin blockade on the accumulation of CD11c-positive cells in the lungs of Flt3L-treated mice. Mice were either injected with vehicle (A–D) or Flt3L for 9 days (10 µg/mouse/d) (E–L) in the presence of either isotype control (A–H) or function-blocking anti-CD11a monoclonal antibody (mAb) (I–L). Subsequently, mice were killed and immunohistochemistry was performed on lung crysections stained with anti-CD11c mAb (B, F, J), anti-CD11b mAb (C, G, K), anti-F4/80 mAb (D, H, L), or control antibody (A, E, I), as indicated. Note that, in anti-CD11a mAb–pretreated mice, numbers of intraalveolar CD11c- and F4/80-positive and CD11b-negative cells representing alveolar macrophages (arrows) remained unchanged, whereas Flt3L-elicited CD11c- and CD11b-positive cells accumulating in the lung interstitial compartment (lung DCs) were strongly reduced. Original magnification, x20.

View larger version (48K): [in this window] [in a new window]   Figure 5. Effect of adhesion blockade versus transient neutropenia on lung dendritic cells (DC) and lung neutrophil (PMN, polymorphonuclear granulocyte) accumulation in Flt3L plus LPS–treated mice. Mice were pretreated with either Flt3L (10 µg/mouse/d for 9 d) or buffer either in the absence or presence of function-blocking antibodies, as indicated, or received an intratracheal application of LPS (1 µg/mouse, 24 h), or were treated with Flt3L for 9 days followed by intratracheal LPS application for 24 hours in the absence or presence of a function-blocking monoclonal antibody (mAb), as indicated. In selected experiments, vehicle- or Flt3L-pretreated mice (9 d) were made transiently neutropenic on Days 8 and 9 of Flt3L application using anti–GR-1 mAb, followed by intratracheal LPS application for 24 hours, as indicated. Subsequently, mice were killed and numbers of lung DCs and lung neutrophils (A) were analyzed in lung homogenates, and numbers of total bronchoalveolar lavage fluid (BALF) cells (B), resident alveolar macrophages (rAM) and alveolar recruited neutrophils (C) as well as alveolar DCs and lymphocytes (Lymph) (D) were determined. Values are presented as mean ± SD of n = 4 animals per group. *Signifies P < 0.05 compared with vehicle-treated mice; $Signifies P < 0.05 compared with Flt3L-only–treated mice; #Signifies P < 0.05 compared with LPS-only–treated mice. itp, isotype control IgG.

Flt3L-elicited Lung DC Accumulation Is Mediated by 2 Integrins

Effect of Flt3L-induced Lung DC Accumulation on the Lung Inflammatory Response to LPS
To evaluate the role of lung DCs in LPS-induced lung inflammation, mice were either left untreated or were pretreated with LPS for 24 hours, or were pretreated with Flt3L for 9 days to increase numbers of lung DCs within the lung parenchymal tissue followed by intratracheal application of LPS for 24 hours. As shown in Figure 5A, LPS application in the absence of Flt3L pretreatment only slightly increased numbers of lung DCs in lung parenchymal tissue, whereas Flt3L application alone significantly increased numbers of lung DCs. However, application of LPS into the lungs of Flt3L-pretreated mice further increased numbers of DCs in lung parenchymal tissue approximately fourfold over lung DC numbers observed in the lungs of Flt3L-only–treated mice. This indicates that Flt3L plus LPS treatment of mice synergistically enhanced the lung DC accumulation compared with either treatment regimen alone. This synergistic action of Flt3L and LPS to elicit a drastic increase in lung DC numbers was strongly and significantly reduced in the presence of anti–CD11a but not anti–CD49d blocking antibodies, again demonstrating that acute inflammatory lung DC accumulation in response to Flt3L plus LPS was critically dependent on engagement of 2 integrins but not 1 integrins (Figure 5A). In addition, the observed corecruitment of neutrophils into the lung parenchymal tissue of Flt3L-treated mice was also further increased in mice challenged with Flt3L plus LPS (Figure 5A). In striking contrast to the inflammatory lung DC accumulation, neutrophil recruitment did not depend on engagement of CD11a or CD49d, corresponding to previously published results (20). Collectively, these data show that both the lung DC and neutrophil accumulation is strongly increased in mice cotreated with Flt3L in the presence of intratracheal LPS, and blockade of CD11a selectively and significantly attenuates the inflammatory lung DC but not lung neutrophil recruitment. Thus, anti–CD11a blocking antibody application is an effective tool to dissect the effects of Flt3L-elicited lung DCs versus lung neutrophils on the lung inflammatory response to endotoxin challenge.

To further differentiate the contribution of Flt3L-elicited lung DCs versus neutrophils on LPS-induced lung inflammation, both Flt3L- and vehicle-pretreated mice were concomitantly depleted of circulating neutrophils via systemic application of anti–GR-1 mAbs. This treatment regimen has been shown recently to deplete circulating neutrophils but not Gr-1–positive monocyte subsets in peripheral blood (14). Importantly, transient neutropenia induced in Flt3L plus LPS– and LPS-only–treated mice efficiently depleted lung neutrophils without affecting numbers of lung DCs (Figure 5A).

Analysis of BALF cellular constituents from mice of the various treatment groups showed that LPS treatment alone elicited a moderate neutrophilic alveolitis with low numbers of corecruited alveolar DCs in the absence of lymphocytes (Figures 5B–5D). Of note, Flt3L treatment alone did not induce an alveolar as opposed to a lung interstitial accumulation of neutrophils or an alveolar DC or lymphocyte recruitment (Figures 5A–5D). In striking contrast, Flt3L-pretreated mice challenged with LPS for 24 hours developed a significantly increased neutrophilic alveolitis compared with mice challenged with LPS alone and recruited significantly more DCs into the alveolar airspace (200–300-fold above control) than was induced by either treatment regimen alone (Figures 5C and 5D). Anti-CD11a but not anti-CD49d application in Flt3L-pretreated mice significantly attenuated the alveolar DC recruitment in response to LPS and also reduced the numbers of alveolar recruited neutrophils (Figure 5C), which was not observed for the lung interstitial compartment (Figure 5A), implying that the 2 integrin CD11a is critical to lung parenchymal and alveolar recruitment of both lung and alveolar DCs, whereas CD11a appears to be more relevant for the neutrophil recruitment across the alveolar epithelial as opposed to the capillary endothelial barrier.

We also assessed the proinflammatory mediator release and lung barrier dysfunction in mice of the various treatment groups. LPS challenge but not Flt3L pretreatment of mice elicited a weak alveolar liberation of TNF- and IL-12 together with a moderate increase in lung permeability, as assessed by increased levels of total protein in the BALF (Figures 6A–6C). In contrast, Flt3L-pretreated and LPS-challenged mice responded with strongly increased BALF TNF- levels (Figure 6A), excessive IL-12 levels in BALF (Figure 6B), and strongly and significantly increased lung permeability, as demonstrated by increased total BALF protein (Figure 6C) and increased leakage of FITC albumin (Figure 6D). This effect could be nearly completely abrogated by pretreatment of the mice with anti–CD11a but not anti–CD49d blocking antibodies (Figures 6A–6D). In contrast, induction of transient neutropenia in mice pretreated with Flt3L plus LPS only weakly attenuated BALF TNF- and IL-12 levels and had no effect on the lung permeability in these mice. Collectively, these data show that LPS challenge of Flt3L-pretreated mice significantly aggravates the lung inflammatory response, as reflected by strongly increased lung parenchymal and alveolar DC and neutrophil numbers, as well as by significantly increased BALF TNF-, IL-12, and serum protein levels. Importantly, selective inhibition of Flt3L-elicited lung parenchymal and alveolar DC accumulation significantly attenuated both the LPS-induced cytokine release and lung permeability changes, whereas transient neutropenia did not attenuate the increased lung permeability observed in Flt3L-pretreated plus LPS-challenged mice.

View larger version (55K): [in this window] [in a new window]   Figure 6. Proinflammatory mediator release and lung barrier dysfunction in FLt3L-pretreated mice challenged with LPS. Mice were left untreated or received function-blocking antibodies, as indicated, together with Flt3L for 9 days either with or without a subsequent LPS challenge. Subsequently, mice were killed and bronchoalveolar lavage (BAL) was performed for determination of BAL fluid (BALF) tumor necrosis factor (TNF)- (A) and IL-12 levels (B), and total protein contents indicative of lung permeability changes (C). Values are presented as mean ± SD of n = 4 animals per group. (D) In addition, in a separate set of experiments, lung permeability changes were assessed by intravenous injection of fluorescein isothiocyanate (FITC)–labeled albumin and subsequent fluorimetric determination of the BALF/serum FITC fluorescence ratio (n = 3). (E) To determine whether pretreatment with Flt3L changed the response of lung dendritic cells (DC) to LPS, lung DCs from both vehicle-treated mice and Flt3L-treated mice were flow-sorted and subsequently stimulated in vitro with 100 ng/ml LPS for 6 hours, followed by TNF-, macrophage inflammatory protein-2 (MIP-2), and IL-12 p35 gene expression analysis using real-time reverse transcriptase–polymerase chain reaction. Data are displayed as fold-change of mRNA levels in LPS-stimulated cells as compared with unstimulated cells. Note that lung DCs from both vehicle-treated mice and Flt3L-pretreated mice responded with similar LPS-stimulated TNF- and MIP-2 mRNA levels in vitro, whereas IL-12 p35 mRNA levels were significantly higher in lung DCs from Flt3L-pretreated mice. Values are given as mean ± SD of n = 3 animals per group. *Indicates P < 0.05 as compared with vehicle-treated mice; #Signifies P < 0.05 compared with LPS-only–treated mice. Isotp = isotype control IgG; mAb = monoclonal antibody.

View larger version (68K): [in this window] [in a new window]   Figure 7. Inflammatory phenotype in a Klebsiella pneumoniae infection model. Mice were pretreated with either Flt3L or vehicle as described for 9 consecutive days. On Day 9, mice were infected intratracheally with 104 cfu K. pneumoniae. (A) Survival of vehicle-treated and Flt3L-treated mice after intratracheal infection with K. pneumoniae (n = 6/group). To analyze the inflammatory phenotype after K. pneumoniae infection, mice were killed at the indicated time points, and bronchoalveolar lavage fluid (BALF) of Flt3L-treated and vehicle-treated mice was analyzed for total cell number (B), dendritic cells (DC) (C), and polymorphonuclear granulocyte (PMN) counts (D), and tumor necrosis factor (TNF)- (E) and IL-12 (F) levels. Lung leakage was assessed using the fluorescein isothiocyanate (FITC) albumin leakage assay (G). Values are given as means ± SD of n = 4–6 animals per group. #Signifies P < 0.05 compared with K. pneumoniae–infected, vehicle-treated mice.

LPS-induced Peritonitis Is Not Amplified by Flt3L Pretreatment
To analyze whether the observed amplification of lung inflammatory responses in Flt3L-pretreated mice is a lung-specific response or might also develop in other organ systems, we challenged both Flt3L- and vehicle-treated mice with an intraperitoneal injection of 20 µg ultrapure LPS, and mice were analyzed 24 hours later. As shown in Figure 8, Flt3L pretreatment led to a strong and significant increase in peritoneal DCs by a factor of approximately 15 and a slight but significant increase in neutrophil counts. In both Flt3L- and vehicle-treated mice, intraperitoneal LPS injection induced a significant increase in neutrophil numbers. However, in marked contrast to the observations made in the lung, Flt3L pretreatment did not provoke increased numbers of DCs or PMNs accumulating in the peritoneal cavity upon intraperitoneal LPS application. Likewise, cytokine levels in the peritoneal lavage fluid were not significantly increased upon Flt3L pretreatment (data not shown), in striking contrast to the observations made in the lung.

View larger version (10K): [in this window] [in a new window]   Figure 8. Peritoneal inflammation after LPS instillation with and without Flt3L pretreatment. Mice were pretreated with either Flt3L or vehicle for 9 days as described. On Day 9, they received an intraperitoneal injection of 20 µg ultrapure LPS or vehicle, as indicated. Peritoneal lavage was performed 24 hours later, and cell numbers and differential cell counts were assessed using Pappenheim-stained cytospins and flow cytometric analysis. Values are presented as mean ± SD of n = 4 animals per group. *Signifies P < 0.05 compared with vehicle-treated mice; #Signifies P < 0.05 compared with LPS-only–treated mice. PMN = polymorphonuclear granulocyte.

	DISCUSSION

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Systemic treatment of mice with Flt3L has been reported to particularly increase numbers of lung myeloid DCs and lung neutrophils (7, 9). The current study confirms and expands previous reports by demonstrating that the Flt3L-elicited lung DC accumulation both in the absence and presence of coapplied LPS is mediated by the 2 integrin CD11a but not the 1 integrin CD49d. Of note, numbers of both lung parenchymal and alveolar accumulating DCs and neutrophils were synergistically increased in Flt3L-pretreated mice cochallenged with LPS, and blockade of CD11a almost completely attenuated the lung parenchymal and alveolar accumulation of myeloid DCs while leaving the concomitant neutrophilic response largely unaffected. Notably, recent studies from our group demonstrated that monocyte recruitment to acutely inflamed mouse lungs was mediated by CD11a, CD11b, and VLA-4, whereas corecruited neutrophils in that model were recruited via CD11b but not CD11a or VLA-4 (20). In addition, recent publications demonstrated an important role of CD11a but not CD11b for the transmigration of both CD11b⁺ CD8^– myeloid DCs, and CD11b^– CD8⁺ lymphoid DCs over resting and TNF-–stimulated endothelial cells in vitro (21), collectively demonstrating an important role of CD11a in the inflammatory myeloid DC recruitment process. Notably, anti-CD11a treatment in Flt3L plus LPS–treated mice did not affect the lung parenchymal neutrophil recruitment but rather reduced the transepithelial migration of elicited neutrophils, together with demonstrating strongly decreased TNF- protein levels and heavily reduced lung permeability. Although neutrophils per se are well accepted to mediate lung permeability changes in various models of acute lung inflammation (22, 23), the present data strongly support the concept that, in addition to neutrophils, lung DCs may also play a critical role in determining the lung barrier integrity in response to acute lung inflammation. This concept is supported by two central observations: First, transient neutropenia was highly effective in depleting both lung parenchymal and alveolar accumulating neutrophils, yet both proinflammatory mediator release and lung permeability in response to Flt3L plus LPS challenge were not affected, indicative of a minor role of lung and alveolar neutrophils in induction of lung permeability in the present model. In contrast, anti-CD11a treatment of the mice to block lung parenchymal and alveolar DC accumulation in response to Flt3L plus LPS was highly effective in reducing BALF TNF- and IL-12 levels and lung permeability changes. Furthermore, both the TNF- and IL-12 release and the lung permeability changes in mice without Flt3L pretreatment did not differ between neutropenic and control mice. Collectively, to the best of our knowledge, the presented findings define a novel, hitherto unrecognized role of lung DCs in regulating the lung barrier integrity in response to bacterial toxins. As such, the present data provide a possible explanation for the clinical observation that critically ill, neutropenic patients are known to develop adult respiratory distress syndrome, a clinical complication associated with a severe loss of lung barrier integrity (24–26).

Although we found that lung DCs do sense bacterial toxins within the alveolar airspace, as demonstrated by increased TNF- and MIP-2 mRNA levels in the sorted lung DC preparations of LPS-challenged mice, we did not find support that Flt3L directly modulates this LPS responsiveness of lung DCs to produce these two classical proinflammatory cytokines, because sorted lung DCs from Flt3L-treated mice had similarly increased TNF- and MIP-2 mRNA levels compared with lung DCs collected from vehicle-only–treated mice. In marked contrast, the capability of lung DCs to produce IL-12 p35 was significantly higher in Flt3L-pretreated as compared with vehicle-treated mice. However, the synergistic effect of Flt3L plus LPS treatment on the lung DC pool and the concomitant lung barrier dysfunction was noted within a 24-hour post–LPS application period, and because Flt3L pretreatment of mice is known to increase numbers of circulating monocytes acting as precursor cells for myeloid DC, it appears likely that Flt3L-elicited lung parenchymal DCs upon LPS challenge additionally promote a rapid mobilization of circulating myeloid DC precursor cells into both the lung parenchymal and alveolar airspace, which ultimately contribute to an aggravated lung inflammatory response.

Recent studies have addressed the effect of Flt3L treatment as an immunotherapeutic option to antagonize chronic or systemic infections in tuberculosis and L. monocytogenes infection models, respectively, in which Flt3L treatment has shown diverse effects (10, 11). To the best of our knowledge, the current study is the first to evaluate the role of lung DCs in a model of acute lung inflammation. Cumulatively, the data show that the 2 integrin–dependent, Flt3L-elicited accumulation of DCs in the lung parenchymal compartment triggers an acute lung injury upon both E. coli endotoxin and infection with K. pneumoniae, which is characterized by an increased neutrophilic alveolitis, proinflammatory mediator release, and high increase in lung permeability. Thus, immunoregulatory or tolerogenic strategies modulating the numbers and/or functions of DC pools to optimize immune responses of the lungs to inhaled inflammatory or infectious antigens need to reflect the considerable proinflammatory potential associated with the lung myeloid DC pool.

	Acknowledgments

 
The expert technical assistance of Emma Braun, Petra Janssen, Margaretha Lohmeyer, and Gudrun Biemer is gratefully acknowledged. The authors thank Dr. Katrin Ahlbrecht for help with the preparation of lung tissue cryosections.

	FOOTNOTES

 
Supported by the German Research Foundation, grant SFB 547 "Cardiopulmonary Vascular System," and the National Network on Community-acquired Pneumonia (CAPNETZ). W.v.W. is supported by a scientific fellowship from Altana Pharma AG, Konstanz, Germany.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200608-1068OC on August 9, 2007

Conflict of Interest Statement: W.v.W. has received a scientific fellowship from Altana Pharma AG, Konstanz, Germany. M.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.M.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. W.S. receives grant and contract support from the following companies: Schering AG, Pfizer Ltd., Altana Pharma AG, Lung Rx, and Myogen. T.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. U.A.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 1, 2006; accepted in final form August 6, 2007

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