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* Department of Pulmonary Medicine, Laboratory for Experimental Lung Research, and
Department of Pathology, Hannover School of Medicine, Hannover, Germany;
Department of Internal Medicine, University of Regensburg, Regensburg, Germany;
Department of Microbiology, University of Alabama, Birmingham, AL 35294; and
¶ School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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, IL-6, keratinocyte derived cytokine, MIP-2, CCL2) and neutrophilic alveolitis upon infection with S. pneumoniae as did control mice with a normal lung DC pool. Unexpectedly, however, Flt3L-pretreated mice, but not control mice, infected with S. pneumoniae developed vasculitis and increased lung permeability by days 2–3 postinfection, and florid pneumonia accompanied by sustained increased bacterial loads by days 3–4 postinfection. This was associated with an overall increased mortality of 
35% by day 4 after pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block inflammatory monocyte-dependent lung mononuclear phagocyte mobilization significantly reduced the lung leakage, but not vasculitis in Flt3L-pretreated mice infected with S. pneumoniae, without affecting the intra-alveolar cytokine liberation or the concomitantly developing neutrophilic alveolitis. Together, the data demonstrate that previous Flt3L-induced lung DC accumulation is not protective in lung innate immunity to challenge with S. pneumoniae, and support the concept that CCR2-dependent mononuclear phagocyte as opposed to neutrophil recruitment contributes to increased lung leakage in Flt3L-pretreated mice challenged with S. pneumoniae. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Streptococcus pneumoniae is the most prevalent pathogen causing community-acquired pneumonia, septic meningitis, and otitis media worldwide (5). S. pneumoniae-derived cytotoxic virulence factors such as pneumolysin are known to induce apoptosis in lung sentinel cells such as alveolar macrophages, and at the same time exert strong cytotoxicity toward alveolar epithelial cells, thereby causing severe lung edema and invasive pneumococcal disease (6, 7). We most recently showed that inflammatory mononuclear phagocyte mobilization is critical to the resolution/repair phase in mice infected with S. pneumoniae (8). Consequently, experimentally increased numbers of mononuclear phagocyte subsets, including myeloid DC in the lungs of mice, improved protective innate immunity to challenge with S. pneumoniae (9). However, no data are currently available that specifically addressed the role of lung myeloid DC in the pathogenesis of pneumococcal lung infection. Because in the lung DC are situated in close vicinity to both capillary endothelial and alveolar epithelial cells, where they form an interdigitating network of sentinel cells specialized to sample inhaled bacterial pathogens (10, 11), it appears conceivable that inhaled pneumococci would have to overcome DC-based immunosurveillance of the lung to cause invasive disease progression. Therefore, in the current study, we hypothesized that mice exhibiting a pre-enriched DC pool in their lungs would respond with an improved protective innate immunity to challenge with S. pneumoniae. To test this hypothesis, mice were pretreated with human rFlt3L to increase their lung DC pool size, and were then infected intratracheally with S. pneumoniae.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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BALB/c mice used in the current study were purchased from Charles River Laboratories and were kept under conventional conditions with free access to food and water. Mice were used in all experiments at 8–12 wk of age in accordance with the guidelines of our Institutional Animal Care and Use Committee. Animal experiments were approved by our local government authorities.
Reagents
Abs used for FACS-based leukocyte subset-specific analysis were purchased from BD Biosciences or Serotec. In selected experiments, the rat anti-mouse anti-CCR2 mAb MC21 was used to block inflammatory mononuclear phagocyte immigration into the lungs of S. pneumoniae-infected mice (50 µg/mouse/day) (12, 13). Flt3L was a gift from Amgen (Thousand Oaks, CA).
Culture and quantification of S. pneumoniae
For infection studies, we used a pneumolysin-producing clinical isolate of capsular group 19 S. pneumoniae (EF3030). The bacteria were grown in Todd-Hewitt broth (Difco) supplemented with 0.1% yeast extract to mid-log phase, and aliquots were snap frozen in liquid nitrogen and stored at –80°C until use, as outlined in detail recently (8, 9, 14). For quantification of pneumococci, serial dilutions of the bacteria were plated on sheep blood agar (BD Biosciences) and incubated at 37°C/5% CO2 for 18 h, followed by the determination of CFU.
Application of Flt3L and infection of mice with S. pneumoniae
Mice were either pretreated with s.c. injections of Flt3L (10 µg/mouse) or pretreated with vehicle (saline) for 9 consecutive days. Subsequently, Flt3L- or vehicle-pretreated mice were infected with S. pneumoniae EF3030 using freshly prepared dilutions of thawed aliquots adjusted to 2.5 x 107 CFU/mouse, as recently described (8). Briefly, tracheas were exposed by surgical resection, and intratracheal instillation of the pneumococci was performed under stereomicroscopic control (Leica Microsystems MS 5) under a laminar flow hood using a 26-gauge catheter (Abbocath; Abbott Laboratories) inserted into the trachea. After instillation, the neck wound was closed with sterile sutures, and mice were kept under specific pathogen-free conditions with free access to autoclaved food and water and were monitored twice daily for disease symptoms during the entire observation period.
Bronchoalveolar lavage (BAL) and lung tissue homogenization for the determination of bacterial loads in the lungs of Flt3L-pretreated and control mice challenged with S. pneumoniae
Bacterial loads within the lungs of S. pneumoniae-infected Flt3L-pretreated and control mice were determined from both whole lung washes and lung tissue homogenates. Briefly, mice were euthanized with an overdose of isoflurane (Forene; Abbott Laboratories), and tracheas of the mice were exposed and cannulated with a shortened 20G needle that was firmly fixed to the trachea. Subsequently, 300-µl aliquots of ice-cold sterile PBS were instilled, followed by careful aspiration until a BAL fluid volume of 1.5 ml was collected. Subsequently, BAL was continued until an additional BAL fluid volume of 4.5 ml was collected. The 1.5- and 4.5-ml BAL fluids (whole lung washes) collected from uninfected or S. pneumoniae-infected mice with or without Flt3L pretreatment were immediately processed for determination of bacterial loads by plating 100 µl of the respective BAL fluid aliquots in 10-fold serial dilutions on sheep blood agar plates, followed by incubation of the plates at 37°C/5% CO2 for 18 h. Subsequently, CFU were counted and bacterial loads in whole lung washes were calculated. Whole lung washes were further subjected to centrifugation at 1400 rpm (4°C, 10 min), and cell pellets were pooled to determine total numbers of BAL fluid leukocytes. In addition, BAL fluid cytokines were measured in cell-free BAL fluid supernatants of the respective 1.5-ml BAL fluid aliquots. Subsequent to BAL, lung and spleen tissue, respectively, were homogenized in 2 ml of HBSS without supplements using a tissue homogenizer (IKA, Staufen, Germany), and 10-fold serial dilutions of lung and spleen tissue homogenates were plated on sheep blood agar plates, followed by incubation of the plates at 37°C/5% CO2 for 18 h and subsequent determination of the CFU.
Quantification and characterization of peripheral blood monocytes from Flt3L-pretreated and control mice
Anticoagulated peripheral blood was collected from vehicle-treated and Flt3L-pretreated mice from the inferior vena cava. Thoroughly mixed peripheral blood was used for preparation of Pappenheim-stained blood smears, and defined volumes of peripheral blood (500 µl per mouse) were subjected to two cycles of RBC lysis in ammonium chloride solution (2 x 5 min, room temperature) and a subsequent determination of total leukocyte counts. Numbers of peripheral blood monocytes in vehicle-treated or Flt3L-pretreated mice were calculated by determining the percentage of monocyte counts in Pappenheim-stained peripheral blood smear preparations and multiplication of those values with the respective white blood cell counts. FACS analysis of peripheral blood monocytes was done by staining blood leukocyte samples with PE-conjugated anti-CD115 and allophycocyanin-conjugated F4/80 Abs, and PerCP-Cy5.5-conjugated anti-Gr-1 Abs together with secondary FITC-labeled anti-CCR2 Ab MC21.
Quantification of alveolar and lung leukocyte subsets
The quantification of alveolar recruited neutrophils contained in BAL fluids of vehicle-treated or Flt3L-pretreated mice either left uninfected or infected with S. pneumoniae was done on differential cell counts of Pappenheim-stained cytocentrifuge preparations, using overall morphologic criteria, including cell size and shape of nuclei and subsequent multiplication of those values by the respective absolute BAL cell counts. Quantification of resident and recruited mononuclear phagocyte subsets contained in BAL fluids recovered from the lungs of control mice and Flt3L-pretreated mice in the absence or presence of pneumococcal infection (BAL fluid alveolar macrophages and exudate mononuclear phagocytes) was done using FACS-based differences in cell surface Ag expression profiles (CD11b, CD11c, F4/80, MHCII, CD86), as outlined in detail recently (8, 9, 15).
Quantification of mononuclear phagocyte subsets in lung parenchymal tissue of uninfected as well as S. pneumoniae-infected Flt3L-pretreated and vehicle-treated mice was done according to recently published protocols (8, 9). Briefly, after BAL, lungs were carefully perfused in situ via the right ventricle with HBSS until lung lobes were visually free of blood. Subsequently, lung lobes were carefully removed while avoiding contaminations with lymphatic tissue or conducting airways, and then cut into small pieces and incubated in digestion solution consisting of RPMI 1640 supplemented with collagenase A and DNase I for 90 min at 37°C. A further disruption of the tissue was done by pipetting with a 1-ml syringe and then passing the lung digests through 100 and 40 µM cell strainers (BD Biosciences), upon which digestion was stopped by adding RPMI 1640/10% FCS. CD11c-positive leukocyte subsets including lung macrophages and lung myeloid DC contained in lung homogenates were further purified using a CD11c MACS kit following the instructions of the manufacturer (Miltenyi Biotec). Briefly, the cells were spun at 1200 rpm for 10 min at 4°C, and the pellet was resuspended in MACS buffer. After a brief centrifugation step, the cells were incubated with octagam (Octapharma) (10 µl of octagam/107 cells) on ice for 10 min to block nonspecific Ab binding, and then washed with MACS buffer and incubated with CD11c beads (10 µl of beads/107 cells) for 15 min at 4°C. After incubation, cells were washed, centrifuged, and passed over a MACS MS column that was gently flushed with MACS buffer to purify the CD11c-positive cells (90% purity). Purified CD11c-positive cells from lung parenchymal tissue digests were then subjected to FACS analysis of differential autofluorescence and cell surface Ag expression profiles, as outlined below, and percentages of mononuclear phagocyte subsets were multiplied by the respective total cell counts of purified CD11c-positive cells.
Immunophenotypic analysis of mononuclear phagocyte subsets in BAL and lung parenchymal tissue
Mononuclear phagocyte subset populations contained in the lung parenchymal tissue of uninfected or S. pneumoniae-challenged control mice and Flt3L-pretreated mice were subjected to flow cytometric immunophenotypic analysis of their cell surface Ag expression profiles. Cells preincubated with octagam were stained for 15 min at 4°C with various combinations of appropriately diluted fluorochrome-conjugated mAbs with specificities for the following cell surface molecules: PE-Cy7-conjugated anti-CD11b, PE-Cy5.5-conjugated anti-CD11c, PE-conjugated anti-CD86, PE-conjugated anti-MHC class II Ab (all from BD Biosciences), and allophycocyanin-conjugated anti-F4/80 (Serotec), as recently described (15). Subsequently, cells were washed in PBS/0.1% BSA/0.02% Na-azide, and cell acquisition was performed on a BD FACS Canto flow cytometer (BD Biosciences) equipped with an argon ion laser operating at 488 nm excitation wavelength and a helium neon laser operating at 633 nm excitation wavelength. Gating of the respective mononuclear phagocyte subsets was done according to their forward light scatter (FSC)-A vs side light scatter (SSC)-A characteristics and FSC-A vs green autofluorescence characteristics, followed by hierarchical subgating according to the differential CD11c vs green autofluorescence characteristics (16). Data analysis and careful postacquisition compensation of spectral overlaps between the various fluorescence channels were performed using BD FACSDiva software (BD Biosciences).
Lung permeability assay
To analyze the effect of Flt3L pretreatment on induction of lung permeability in mice challenged with S. pneumoniae, mice received an i.v. injection of FITC-labeled human albumin (1 mg/mouse in 100 µl of saline) (Sigma-Aldrich) 1 h before mice were euthanized by isoflurane. Undiluted BAL fluid samples and serum samples (diluted 1/100 in saline) were placed in a 96-well microtiter plate, and fluorescence intensities were measured using a fluorescence spectrometer (FL 880 microplate fluorescence reader; Bio-Tek Instruments) operating at 488 nm absorbance and 525 ± 20 nm emission wavelengths. The lung permeability index is defined as the ratio of fluorescence signals of undiluted BAL fluid samples to fluorescence signals of 1/100 diluted serum samples (12).
Lung histopathology
Vehicle-treated mice and Flt3L-pretreated mice either were left uninfected or were infected with S. pneumoniae and then killed at various time points postinfection. Subsequently, lungs were inflated in situ with a prewarmed solution of Tissue-Tek (Sakura) kept at 37°C. Thereafter, the lungs were carefully removed and immersed in PBS-buffered formaldehyde solution (4.5% (pH 7.0)) for at least 24-h fixation at room temperature. Lung tissue samples were then paraffin embedded, and lung sections of 5 µm were stained with H&E and Elastica-van-Gieson and examined histopathologically using a Zeiss Axiovert 200 M microscope (Zeiss).
The quantification of vasculitis was done by determining total areas of two sections of the completely embedded lung specimen of either control mice or Flt3L-pretreated mice of the respective treatment groups infected with S. pneumoniae by planimetry. Subsequently, numbers of cross-sections of vessels fulfilling the criteria for vasculitis were counted and expressed per square centimeter of lung tissue. Vasculitis is defined as neutrophilic infiltration of the vessel wall with karyorectic debris and at least segmental necrosis of the medium, sometimes with associated fibrin thrombi.
ELISA
Proinflammatory cytokine release in BAL fluids of uninfected or S. pneumoniae-infected control mice and Flt3L-pretreated mice was determined using commercially available ELISA (R&D Systems).
Statistics
All data are given as mean ± SD. Differences between untreated and Flt3L-pretreated mice infected with S. pneumoniae were analyzed by ANOVA, followed by post hoc Dunnett test. Differences between groups of S. pneumoniae-infected mice pretreated with Flt3L in the absence or presence of MC21 were analyzed by ANOVA, followed by post hoc Scheffe test. Differences between groups were analyzed by Levene’s test for equality of variances, followed by Student’s t test using SPSS for Windows software package. Survival curves were compared by log-rank test. Statistically significant differences between groups were assumed when p values were <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We initially analyzed the effect of Flt3L pretreatment on circulating monocyte and neutrophil counts and the lung myeloid DC pool size in uninfected mice. Vehicle-treated mice were found to have 1.1 ± 0.4 x 102 monocytes and 5.1 ± 2.4 x 102 neutrophils per µl of peripheral blood, whereas Flt3L-pretreated mice had 1.2 ± 0.4 x 103 monocytes (n = 4; p < 0.02) and 1.3 ± 0.8 x 103 neutrophils per µl of peripheral blood (n = 4; NS). Moreover, as shown in Fig. 1, FACS-based characterization of CD11c-positive cells purified from lung parenchymal tissue of vehicle-treated or Flt3L-pretreated mice yielded two major mononuclear phagocyte subsets (P1, P2), as defined by their differential green autofluorescence properties in conjunction with cell surface Ag expression profiles of CD11c, CD11b, MHCII, and CD86, corresponding to recent reports (16). CD11c-positive cells collected from the lung parenchymal tissue of control mice according to their differential autofluorescence and immunophenotype were mainly composed of lung macrophages (P2; Fig. 1A) and, to a lesser extent, of lung myeloid DC (P1; Fig. 1, A, G, and H). FACS analysis of these two major mononuclear phagocyte subsets showed that lung macrophages were green autofluorescent and CD11c+, CD11b–, MHCII–/low, and CD86–, whereas lung myeloid DC lacked green autofluorescence, but were found to be CD11c+ and CD11b+, MHCIIhigh, and CD86+ (P1; Fig. 1, A and C). Of note, Flt3L-pretreated mice had a significantly (14-fold) increased lung myeloid DC pool size with a similar immunophenotypic profile, compared with lung DC of vehicle-treated control mice (P1; Fig. 1, B, D, and H). At the same time, numbers of lung macrophages did not differ significantly between Flt3L-pretreated as compared with vehicle-treated control mice (P2; Fig. 1, A, B, and G). Also, Flt3L pretreatment of mice did not affect numbers of resident alveolar macrophages or alveolar exudate mononuclear phagocytes compared with vehicle-treated controls (Fig. 1, E and F). Finally, both vehicle-treated and Flt3L-pretreated mice had a small, but detectable portion of plasmacytoid DC (CD11clow-mid, CD11b–, B220+) in their lung homogenates (data not shown).
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Initial dose-response experiments revealed that intratracheal challenge of BALB/c mice with 3 x 107 CFU S. pneumoniae provoked an increased mortality of infected mice of 50% by day 3 postinfection (data not shown) (9). Therefore, in the current study, S. pneumoniae infection doses were adjusted to 2.5 x 107 CFU S. pneumoniae/mouse.
Mice pretreated with Flt3L responded with progressive acute lung injury upon infection with S. pneumoniae (2.5 x 107 CFU/mouse), as characterized by a rapidly developing, significantly increased vasculitis and microthrombus formation (Fig. 2A), and significantly increased lung permeability at day 2, peaking at day 3 postinfection, with a further decline by day 7 postinfection (Fig. 2B). As shown in Fig. 2, C and D, H&E-stained lung tissue sections of Flt3L-pretreated mice challenged with S. pneumoniae revealed confluent bronchopneumonia by day 2 postinfection, progressing into lobar pneumococcal pneumonia with a significantly increased degree of inflamed lung tissue of 50% by day 4 postinfection, and resolving thereafter by day 14 postinfection (Fig. 2, C and D). In contrast, control mice infected with 2.5 x 107 CFU S. pneumoniae did not develop increased vasculitis or lung permeability (Fig. 2, A and B) and showed a mild bronchopneumonia not progressing toward lobar pneumonia with an overall inflamed lung tissue not exceeding 10% with a restitutio ad integrum observed by day 8 postinfection (Fig. 2, C and D).
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35% of the Flt3L-pretreated mice, but none of the control mice succumbed to pneumococcal pneumonia (Fig. 3C), collectively demonstrating that Flt3L pretreatment of mice results in a decreased bacterial pathogen elimination process and a severely aggravated lung inflammatory response to Gram-positive bacterial infection. To further investigate whether the increased lung permeability observed in the Flt3L-pretreated mice may have caused septic disease progression in these mice subsequent to pneumococcal infection, we determined bacterial loads in whole blood and spleen homogenates from mice of either treatment group during the observation period of 7 days. Interestingly, none of the investigated mice of either treatment group was found to demonstrate septic disease progression during the observation period, thus excluding sepsis induction as the underlying reason for the decreased survival noted in the Flt3L-pretreated mice (data not shown).
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Analysis of proinflammatory mediator profiles in BAL fluids of Flt3L-pretreated and vehicle-treated, S. pneumoniae-infected mice revealed rapidly increased TNF- protein levels upon infection, peaking by day 1 postinfection with visual differences between groups particularly on day 4 postinfection, although these differences did not reach statistical significance (Fig. 4A). Moreover, mice of either treatment group initially responded with strongly elevated neutrophil chemoattractants MIP-2 and keratinocyte derived cytokine (KC) (CXCL1/2), the monocyte chemoattractant CCL2, and the acute-phase protein IL-6, with peak levels mainly observed between day 1 up until day 3 postinfection (Fig. 4, B–E). Although Flt3L-pretreated mice compared with vehicle-treated mice demonstrated slightly, but significantly elevated MIP-2, KC, CCL2, and IL-6 cytokine levels at day 4 postinfection, overall profiles of cytokine responses between mice of either treatment group were largely similar during the observation period of 7 days.
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It is currently unknown whether Flt3L pretreatment of mice affects phenotypes of circulating monocytes or monocyte-derived mononuclear phagocyte subsets either under baseline or inflammatory conditions in vivo, which may have possible implications for the mobilization of monocyte-derived macrophage subsets and the resolution of inflammation. FACS analysis of blood monocyte subsets of vehicle-treated and Flt3L-pretreated mice revealed that circulating monocytes of either treatment group coexpressed CD115 and F4/80 and were composed of Gr-1+/CCR2+ and Gr-1–/low/CCR2–/low monocyte subsets (Fig. 6, A–D). This demonstrates that Flt3L treatment of mice elicited quantitative rather than qualitative changes in peripheral blood monocyte subset distribution profiles. Moreover, FACS analysis of alveolar macrophages collected from the lungs of vehicle-treated mice (Fig. 6, E and F) and Flt3L-pretreated mice (Fig. 6, G and H) revealed a F4/80+, CD115–, CCR2–/low, and Gr-1– immunophenotypic profile of these cells, with no differences observed between groups (Fig. 6, E–H). A similar immunophenotypic profile was also analyzed on alveolar macrophages recovered from the lungs of both vehicle-treated and Flt3L-pretreated mice challenged with S. pneumoniae for either 4 or 7 days (data not shown in detail). In contrast, alveolar recruited exudate macrophages (P2; Fig. 6, I–M) recovered from the lungs of vehicle- or Flt3L-pretreated mice challenged with S. pneumoniae were found to be CD11b+, CD115–, CCR2+, and Gr-1+/middle (Fig. 6, I and K, middle histogram), with no overt differences in immunophenotypic profiles of alveolar exudate macrophages between treatment groups. These data support the view that Flt3L pretreatment of mice does not affect phenotypic profiles of inflammatory monocytes or monocyte-derived mononuclear phagocyte subsets (alveolar macrophages, exudate macrophages), but rather elicits quantitative changes in baseline (e.g., lung DC) and inflammatory elicited lung mononuclear phagocyte subset recruitment in response to bacterial infection.
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We next questioned whether the significantly increased inflammatory mononuclear phagocyte subset recruitment particularly observed by day 2 postinfection accounted for the increased vasculitis and later developing lung permeability peaking by day 3 postinfection in the Flt3L-pretreated mice challenged with S. pneumoniae. Application of anti-CCR2 Ab MC21, which is known to particularly block inflammatory Gr1+/CCR2+ monocyte subset recruitment (12, 17), nearly completely inhibited peak lung barrier dysfunction (Fig. 7A), but not vasculitis (data not shown) in Flt3L-pretreated mice, which was observed to increase by day 2 and to peak by day 3 postpneumococcal challenge. Importantly, anti-CCR2 Ab application did not affect numbers of alveolar macrophages as opposed to alveolar exudate macrophages, lung macrophages, and lung DC in Flt3L-pretreated and vehicle-treated mice challenged with S. pneumoniae (Fig. 7, B–E). At the same time, numbers of alveolar recruited neutrophils were not affected by MC21 application in Flt3L-pretreated or vehicle-treated mice infected with S. pneumoniae for 3 days (Fig. 7F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Flt3L-pretreated mice challenged with S. pneumoniae exhibited an overall mortality of 35% by day 4 postinfection, as opposed to vehicle-treated control mice not succumbing to pneumococcal infection during the observation period of 7 days. Importantly, this increased mortality in the Flt3L-pretreated mice was preceded by an early developing vasculitis, microthrombus formation, and increased lung permeability by days 2–3 postpneumococcal infection and an impaired capacity of the Flt3L-pretreated mice to purge the pneumococcal infection. Flt3L pretreatment of mice alone was not sufficient to elicit the reported increased vasculitis and lung permeability changes, thus clearly involving an inflammatory activation step. Importantly, whole blood and spleen tissue homogenates collected from infected mice of either treatment group during the observation period of 7 days did not provide evidence for septic disease progression in the currently described Flt3L pneumonia model, thus excluding the possibility that the decreased capacity to purge the pneumococcal infection along with the increased lung permeability observed in the Flt3L-pretreated mice may have accounted, at least in this regard, to the observed decreased survival in these mice. Moreover, proinflammatory cytokine/chemokine levels analyzed in BAL fluids of mice of the two treatment groups during the observation period of 7 days exhibited similar profiles, with only slight differences observed between groups by day 4 postinfection, thus strongly arguing against a major impact of proinflammatory mediators to directly contribute to the noted differences in outcome between treatment groups. In contrast, we found that Flt3L application triggered both a transient peripheral blood neutrophilia and monocytosis in mice, followed by a strongly increased accumulation of myeloid DC in lung parenchymal tissue, in line with recent reports (4). Therefore, it is conceivable that increased numbers of neutrophils and/or inflammatory monocytes or monocyte-derived mononuclear phagocyte subsets such as lung myeloid DC contributed to the vasculitis and/or lung permeability changes as well as decreased pneumococcal clearance capacities, with possible implications for the decreased survival noted in the Flt3L-pretreated mice challenged with S. pneumoniae. Of note, FACS analysis of peripheral blood monocytes collected from vehicle-treated or Flt3L-pretreated mice largely ruled out the possibility that Flt3L pretreatment of mice selectively affected monocyte subset compositions in peripheral blood. In contrast, Flt3L pretreatment strongly increased particularly numbers of lung DC, which are positioned in close vicinity to both capillary endothelium and alveolar epithelium to evoke lung permeability changes upon inflammatory activation. In addition to lung DC acting as possible culprits in the induction of lung permeability in the described model, neutrophils have been implicated as classical effector cells in acute lung injury both in murine models of lung inflammation and in human diseases (18). However, both BAL fluid levels of neutrophil chemoattractants KC and MIP-2 (CXCL1/2) and the concomitantly developing neutrophilic alveolitis observed at day 1 up until day 3 postinfection were not different in vehicle-treated as compared with Flt3L-pretreated, S. pneumoniae-infected mice, thus strongly arguing against a major role of inflammatory recruited neutrophils in induction of lung barrier dysfunction in the Flt3L-pretreated mice infected with S. pneumoniae. In contrast, application of anti-CCR2 Ab MC21 strongly inhibited the lung barrier dysfunction in mice of the Flt3L group without blocking the developing neutrophilic alveolitis or vasculitis, thus again excluding elicited neutrophils to contribute to the lung permeability observed in the current model. Anti-CCR2 Ab MC21 is known to efficiently block inflammatory monocyte recruitment toward acutely inflamed lungs (7, 12, 17, 19). Thus, application of MC21 to block CCR2 function in vivo was expected to inhibit inflammatory mobilization of the CCR2-positive rather than CCR2-negative circulating monocyte subset in Flt3L-pretreated mice upon infection with S. pneumoniae. Indeed, application of anti-CCR2 Ab MC21 significantly reduced the lung barrier dysfunction in Flt3L-pretreated, S. pneumoniae-infected mice, while at the same time strongly inhibiting numbers of monocyte-derived lung parenchymal macrophages, alveolar exudate macrophages, and particularly lung DC. These data strongly support the concept that the significantly increased lung permeability observed in Flt3L-pretreated as compared with vehicle-treated mice infected with S. pneumoniae is most probably due to increased inflammatory lung mononuclear phagocyte subset recruitment in these mice, as a consequence of the Flt3L pretreatment regimen, and may have contributed to the overall reduced outcome in the Flt3L treatment group. However, because inflammatory monocytes have a considerable plasticity to differentiate into lung macrophages and lung myeloid DC (20, 21), and because MC21 application selectively blocks the CCR2-dependent inflammatory monocyte recruitment and therefore derived mononuclear phagocyte subset mobilization, we are currently not able to further pinpoint the observed lung permeability changes noted in the Flt3L-pretreated mice to a specific mononuclear phagocyte subset. Collectively, the data of the current study for the first time support the concept of an important involvement of CCR2-dependent inflammatory mononuclear phagocyte interference with sessile barrier cells of the lung to provoke lung barrier dysfunction and poor outcome in Flt3L-pretreated mice infected with S. pneumoniae.
We recently demonstrated that global deletion or inhibition of signaling pathways with critical relevance for inflammatory mononuclear phagocyte recruitment such as represented by PI3K
and CCR2 may result in detrimental effects on outcome in pneumococcal pneumonia (8, 9). In contrast, CCL2 chemokine-dependent mononuclear phagocyte mobilization toward S. pneumoniae-challenged lungs using CCL2-overexpressing mice improved protective immunity of the lung to challenge with S. pneumoniae, although at the same time eliciting fibroproliferative responses in infected lungs (9). In this context, alveolar macrophages are currently the best-characterized mononuclear phagocyte subset of the lung that mediates central steps of the host defense against inhaled bacterial pathogens, including the phagocytosis and killing of bacteria. Alveolar macrophages that undergo apoptosis in response to S. pneumoniae infection may contribute to enhanced bacterial killing, and additionally restrict the proinflammatory response to inhaled bacteria, thus attenuating overwhelming local and systemic proinflammatory responses to S. pneumoniae infection. At the same time, alveolar and exudate macrophages play important roles in the resolution/repair phase of the disease, thus serving to regain alveolar and lung homeostasis (22, 23, 24, 25, 26). The currently presented data, making use of a largely (yet not exclusively) DC-specific growth factor for the first time, aim at gaining further insights into the role of mononuclear phagocyte subsets other than classical alveolar macrophages in the lung host defense to S. pneumoniae infection and highlight the critical importance of a fine-tuned mononuclear phagocyte network within the lung for an optimal host defense to inhaled bacterial pathogens. In view of future clinical strategies aiming at improving DC-dependent adaptive immune responses to noninfectious diseases, such as metastatic lung cancer (27, 28), it will be important to determine whether these intervention strategies to modulate DC pool sizes interfere with lung inflammatory responses to pulmonary infections.
Collectively, our initial hypothesis that Flt3L pretreatment renders mice more resistant to challenge with the prototype Gram-positive bacterial pathogen, S. pneumoniae, was wrong. The data of the current study show that a Flt3L-induced increase in numbers of lung myeloid DC and possibly exudate macrophages may have serious consequences for the host challenged with S. pneumoniae, thus arguing against therapeutic interventions to modulate numbers of lung DC as a strategy to improve lung innate immunity against invading bacterial pathogens.
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Acknowledgment |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This study has been supported by the German Research Foundation, Deutsche Forschungsgemeinschaft Grant SFB 587 (to U.A.M. and T.W.). 
2 Address correspondence and reprint requests to Dr. Ulrich A. Maus, Department of Pulmonary Medicine, Laboratory for Experimental Lung Research, Hannover School of Medicine, Hannover 30625, Germany. E-mail address: Maus.Ulrich{at}mh-hannover.de
3 Abbreviations used in this paper: Flt3L, FMS-like tyrosine kinase 3 ligand; BAL, bronchoalveolar lavage; DC, dendritic cell; FSC, forward light scatter; KC, keratinocyte derived cytokine; SSC, side light scatter.
Received for publication March 16, 2007. Accepted for publication June 26, 2007.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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in the host defense against pneumococcal infection. Am. J. Respir. Crit. Care Med. 175: 958-966. This article has been cited by other articles:
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  Z. Shao, T. O. Makinde, H. S. McGee, X. Wang, and D. K. Agrawal Fms-Like Tyrosine Kinase 3 Ligand Regulates Migratory Pattern and Antigen Uptake of Lung Dendritic Cell Subsets in a Murine Model of Allergic Airway Inflammation J. Immunol., December 1, 2009; 183(11): 7531 - 7538. [Abstract] [Full Text] [PDF]
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C. Winter, W. Herbold, R. Maus, F. Langer, D. E. Briles, J. C. Paton, T. Welte, and U. A. Maus Important Role for CC Chemokine Ligand 2-Dependent Lung Mononuclear Phagocyte Recruitment to Inhibit Sepsis in Mice Infected with Streptococcus pneumoniae J. Immunol., April 15, 2009; 182(8): 4931 - 4937. [Abstract] [Full Text] [PDF]
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F. Pene, B. Zuber, E. Courtine, C. Rousseau, F. Ouaaz, J. Toubiana, A. Tazi, J.-P. Mira, and J.-D. Chiche Dendritic Cells Modulate Lung Response to Pseudomonas aeruginosa in a Murine Model of Sepsis-Induced Immune Dysfunction J. Immunol., December 15, 2008; 181(12): 8513 - 8520. [Abstract] [Full Text] [PDF]
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J. J. Osterholzer, J. L. Curtis, T. Polak, T. Ames, G.-H. Chen, R. McDonald, G. B. Huffnagle, and G. B. Toews CCR2 Mediates Conventional Dendritic Cell Recruitment and the Formation of Bronchovascular Mononuclear Cell Infiltrates in the Lungs of Mice Infected with Cryptococcus neoformans J. Immunol., July 1, 2008; 181(1): 610 - 620. [Abstract] [Full Text] [PDF]
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in E–H) or were pretreated with Flt3L (10 µg/mouse, s.c.) for 9 consecutive days (B and D; 
in E–H). Subsequently, mice were euthanized, BAL was performed, and CD11c-positive cells were purified from lung parenchymal tissue digests, as described in Materials and Methods. A and B, Representative flow cytometry dot plots of MACS-purified CD11c-positive lung mononuclear phagocyte subsets. Cells were gated according to their FSC vs SSC characteristics, followed by hierarchical subgating using their FSC vs green autofluorescence (FITC) properties. Gated cells (P1, P2) in A and B represent either low green autofluorescent, CD11c-positive lung myeloid DC (P1), or strongly green autofluorescent lung macrophages (P2) of control mice (left dot plots) or Flt3L-pretreated mice (right dot plots). C and D, Gated cells (P1, P2) of control mice (C) or Flt3L-pretreated mice (D) were further subjected to immunophenotypic analysis of their CD11c, CD11b, MHCII, and CD86 cell surface Ag expression profiles, as indicated. E–H, FACS-based quantification of alveolar macrophages (E), exudate mononuclear phagocytes (F), lung macrophages (G), or lung DC (H) collected by either BAL or MACS enrichment of CD11c-positive mononuclear phagocytes of lung tissue digests of either vehicle- or Flt3L-pretreated mice. Values are shown as mean ± SD of n = 6 mice per treatment group. **, Indicates significant increase (p < 0.01) compared with vehicle-treated control mice.
