Published online before print November 24, 2004
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,1
Departments of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine,
* Cell and Developmental Biology, and
¶ Cancer Biology,
Section of Surgical Sciences, Vanderbilt University School of Medicine, and
Department of Veterans Affairs, Nashville, Tennessee
1 Correspondence: Allergy, Pulmonary and Critical Care Medicine, Associate Professor of Cell and Developmental Biology, Vanderbilt University School of Medicine, T-1218 MCN, Nashville, TN 37232-2650. E-mail: timothy.blackwell{at}vanderbilt.edu
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B in response to Eschericia coli lipopolysaccharide demonstrated normal macrophage function. Optimizing this methodology provides an important tool for the study of specific genes and their contribution to alveolar macrophage function in vivo.
Key Words: lung • chimera • green fluorescent protein • mouse • transgenic
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In the lungs, the extent and timing of macrophage reconstitution are not well defined; therefore, we undertook these studies to determine the kinetics of alveolar macrophage reconstitution following FLT. FLT experiments were performed using transgenic donors that constitutively express green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) immediate early enhancer coupled to a ß-actin promoter. After determining the percentage reconstitution of peripheral blood monocytes (PBM) by fluorescence activated cell sorting (FACS), repopulation of alveolar macrophages with donor-derived cells in bone marrow chimeric mice was determined by identifying recipient and donor macrophages by immunohistochemistry on tissue sections and FACS analysis of cells obtained by bronchoalveolar lavage. We reasoned that elimination of residual recipient alveolar macrophages following FLT would accelerate macrophage repopulation with donor-derived cells. To eliminate alveolar macrophages, we administered liposomal clodronate by intratracheal (IT) injection. This treatment has been shown to result in selective apoptosis of macrophages [10 , 11 ]. We administered clodronate at 6 weeks after FLT, a time-point at which PBM were reconstituted, but alveolar macrophages were still predominantly of recipient genotype. In these studies, we found that clodronate treatment accentuated repopulation with phenotypically normal alveolar macrophages.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B-dependent HIV-1 LTR promoter [13
, 14
]. Wild-type (WT) mice (B6;129 background) were obtained from Jackson Laboratories (Bar Harbor, ME).
FLT
FLT experiments were performed as follows: Timed matings were set up with Nagy GFP mice, and females were checked on consecutive days until vaginal plugs were observed. On day E14.5, pregnant females were killed by CO2 inhalation, and the uterus was surgically removed. Fetuses were separated using forceps and placed into culture media (RPMI 1640, Invitrogen, Carlsbad, CA). Embryonic livers were removed with the aid of a dissecting microscope, pooled into an Eppendorf tube containing 1 ml RPMI, and stored on ice. Single-cell suspensions were prepared by drawing cells through needles of decreasing bore size (18, 23, 25 gauge). Recipient mice were lethally irradiated using a 137cesium 
source by giving a split dose of 800 rads followed 3 h later by 400 rads. After irradiation, 2 million donor cells were injected intravenously (i.v.) via tail vein. To diminish infection, mice were maintained on antibiotics (polymyxin B and neomycin) for 2 weeks after FLT and acidified water (pH 2.7) for the duration of the experiment.
Liposomal clodronate preparation and administration
Liposomal encapsulation of clodronate (dichloromethylene diphosphonate) was performed as described previously [15
]. Briefly, a mixture of 8 mg cholesterol (Sigma Chemical Co., St. Louis, MO) and 86 mg egg-phosphatidylcholine (dioleoyl-phosphatidylcholine, Avanti, Alabaster, AL) was dissolved in chloroform and then evaporated under nitrogen. Chloroform was further removed by placing under low vacuum in a speedvac the Savant concentrator(Thermo Electron Corp., Holbrook, NY). The clodronate solution was prepared by dissolving 1.2 g dichloromethylene diphosphonic acid (Sigma Chemical Co.) in 5 ml sterile phosphate-buffered saline (1x PBS). The clodronate solution (5 ml) was added to the liposome preparation and mixed thoroughly. The solution was sonicated and ultracentrifuged at 10,000 g for 1 h at 4°C. The liposome pellet was removed, resuspended in 5 ml PBS, and ultracentrifuged at 10,000 g for 1 h at 4°C. Liposomes were removed, resuspended in 5 ml PBS, and used within 48 h. The final concentration of the liposomal clodronate solution was 5 mg/ml. A single dose of liposomal clodronate was administered via IT injection 6 weeks following FLT.
FACS
Peripheral blood samples (0.5 ml) were collected and placed in a polystyrene tube containing 0.1 ml 0.5 M EDTA (pH 8.0) at room temperature. The samples were washed with 3 ml 1x FACS buffer (1xPBS with 1% bovine serum albumin) and centrifuged at 300 g for 15 min at room temperature. After resuspending in 50 µl 1x PBS, 4 µl Fc block (PharMingen, San Diego, CA) was added and stored on ice for 10 min. To label monocytes, 2 µl CD11b-phycoerythrin (PE) and 2 µl Gr-1-allophycocyanin (APC; PharMingen) were added and stored on ice for 10 min. Lysis buffer (10 mL; 8.29 g NH4Cl, 1 g KHCO3, 37.2 mg Na2EDTA per liter H2O, pH 7.4) was added to lyse red blood cells. Subsequently, cells were centrifuged at 300 g for 15 min at room temperature, washed with 5 ml FACS buffer, and resuspended in 3 ml FACS buffer for analysis. FACS was then performed using standard protocols using a Becton Dickinson (San Jose, CA) FACScan flow cytometer. For bronchoalveolar lavage, samples were collected in saline and placed on ice. Lavage cells were pelleted at 300 g for 15 min. After resuspending in 50 µl 1x PBS, 4 µl Fc block (PharMingen) was added and stored on ice for 10 min. To label macrophages, 2 µl CD11b-PE (PharMingen) was added and stored on ice for 10 min. Cells were then washed with 1x PBS and resuspended in 200 µl FACS buffer for analysis.
Lung immunohistochemistry and macrophage counting
To collect lung tissue, mice were perfused with saline, and lungs were inflated with 1 ml 10% neutral-buffered formalin (NBF). Lungs were stored in NBF overnight at 4°C and then paraffin-embedded. Sections (5 µ) were cut and placed on charged slides. Following paraffin removal, sections were rehydrated and placed in heated target retrieval solution, high pH (DakoCytomation, Carpinteria, CA) for 20 min. Endogenous peroxidase was quenched with 0.03% hydrogen peroxide, and samples were treated with casein-based protein-blocking solution (DakoCytomation) prior to primary antibody addition. Tissues were incubated with goat anti-CD68 (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-GFP (Clontech, Palo Alto, CA) for 30 min. Sections without primary antibody served as negative controls. The Vectastain ABC Elite (Vector Laboratories, Burlingame, CA) system and diaminobenzidine (DAB)+ (DakoCytomation) were used with CD68 antibodies, and rabbit Envision+ system, horseradish peroxidase/DAB+ (DakoCytomation) was used with GFP antibodies to produce localized, visible staining. Slides then were lightly counterstained with Mayer’s hematoxylin, dehydrated, and coverslipped.
Quantification of total alveolar macrophages and GFP-positive alveolar macrophages was performed on serial sections immunostained with GFP and CD68 antibodies. Sections were visualized under 400x magnification, and digital pictures of 10 serial, nonoverlapping fields were taken using Magnifire SP software (Optronics, Goleta, CA). The number of GFP-positive and CD68-positive cells per field was recorded. The results were verified by blinded analysis from a second observer.
Pseudomonas aeruginosa administration
P. aeruginosa (strain PA103) was streaked onto trypticase soy agar plates and grown in deferrated dialysates of tripticase soy broth supplemented with 10 mM nitrilotriacetic acid (Sigma Chemical Co.), 1% glycerol, and 100 mM monosodium glutamate at 33°C for 1–3 h in shaking incubator. Cultures were centrifuged at 8500 g for 5 min, and the bacterial pellet was washed twice in Ringers lactate. The bacteria were diluted to the appropriate number of colony forming units (CFU) per ml in Ringers lactate solution as determined by spectrophotometer. The bacterial concentration was confirmed by diluting the samples and plating the known dilution on sheep blood agar plates. Pseudomonas (106 CFU) was administered by IT injection.
P. aeruginosa lung colony counts
The lungs were removed aseptically and placed in 3 ml sterile saline. The lungs were then homogenized in a tissue homogenizer in a vented hood under sterile conditions. Serial dilutions of the homogenates were prepared, and 10 µl each dilution was plated in soy-based blood agar plates (BD, Sparks, MD). The plates were incubated for 18 h at 37°C, and the number of colonies was counted and recorded.
Bioluminescence imaging
Mice were anesthetized and shaved over the chest before imaging. Luciferin (1 mg/mouse in 100 µl isotonic saline) was administered by i.v. injection, and mice were imaged with an ultrasensitive charge-coupled device (CCD) camera (IVIS Imaging System, Alameda, CA). For the duration of photon counting, mice were placed inside a light tight box that housed the camera. Light emission from the mouse was detected as photon counts by the ultrasensitive CCD camera and customized image processing hardware and software (Living Image Software, Alameda, CA). The imaging duration (15 s) was selected to avoid saturation of the camera during image acquisition. Quantitative analysis was performed by a defining standard area over the mid-lung zone and determining the total integrated photon intensity over the area of interest.
Lipopolysaccharide (LPS) administration
Gram-negative Escherichia coli LPS (Serotype 055:B5) was obtained from Sigma Chemical Co. A working solution of LPS was made by resuspending 1 mg LPS in 10 ml sterile PBS. For IT injections, a single dose of 75 µg LPS was administered.
Statistical analysis
To assess differences among groups, analyses were performed with GraphPad Instat (GraphPad Software, San Diego, CA) using a one-way ANOVA test (Pvalues <0.05 were considered significant).
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Figure 1. FACS analysis of peripheral blood cells from a Nagy GFP transgenic mouse showing (A) selection of PBM population by forward- and side-scatter characteristics, (B) analysis of population from A showing cells are positive for CD11b-PE and Gr-1-APC, and (C) identification of dual-positive cells (CD11b and Gr-1), which are also GFP-positive.
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Figure 2. Kinetics of PBM reconstitution following FLT with GFP+ donor cells. CD11b/Gr-1 dual-positive monocytes were analyzed by FACS to determine the percentage that were GFP+ (donor genotype) in the following groups: WT mice, chimeric mice 4 weeks after FLT with GFP+ donor cells (4 Week), chimeric mice 10 weeks after FLT with GFP+ donor cells (10 Week), and chimeric mice 10 weeks after FLT + treatment with liposomal clodronate (10 Week + Clod). Each bar represents the mean ± SEM of seven mice per group.
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We evaluated lung sections to determine the extent of repopulation of resident alveolar macrophages with GFP+ donor cells after FLT. GFP+ donor cells were identified in the lungs of chimeric mice by fluorescence microscopy. In Nagy GFP controls that did not undergo FLT, distinct GFP+ cells, primarily alveolar macrophages as identified by morphology and location, were visible (Fig. 3A ). No brightly green fluorescent cells were observed in untransplanted WT controls (Fig. 3B) . At 10 weeks after FLT, green fluorescent cells with the characteristic appearance of macrophages were observed in the lungs (Fig. 3C) ; however, increased numbers of GFP+ cells were observed in lung sections of clodronate-treated mice at 10 weeks following FLT (Fig. 3D) . To better quantify the extent of alveolar macrophage repopulation, immunohistochemistry was used to identify all alveolar macrophages using anti-CD68 antibodies [17 ] and cells of donor origin using anti-GFP antibodies. Serial sections were stained, and alveolar macrophage repopulation was quantified by counting CD68+ and GFP+ macrophages in 10 high-power fields of lung parenchyma per slide (Fig. 4 ). Figure 5 illustrates the number of CD68+ and GFP+ macrophages by treatment group. CD68 immunostaining revealed no significant differences in the number of macrophages in untreated Nagy GFP and WT controls. Compared with these controls, CD68+ macrophage numbers were similar at 4 or 10 weeks after FLT (with or without clodronate treatment). GFP+ macrophages were not identified in untransplanted WT controls, and at 4 weeks after FLT, only 1.4 ± 0.4 cells per field were identified as immunoreactive for GFP, representing 9.5% of total macrophages. By 10 weeks after FLT, however, there were 6.8 ± 0.4 GFP+ macrophages per field, representing 48% of total macrophages. Chimeric mice treated with clodronate displayed a significant increase in GFP+ macrophages (14.4±0.5, representing 90% of total macrophages) as compared with chimeric mice without clodronate treatment at 10 weeks (P<0.001). No other cell types in the lung were observed to be GFP+ at any time-point after FLT.
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Figure 3. Fluorescence microscopy of lung tissue sections showing GFP+ cells in the following: (A) Nagy GFP transgenic mouse, (B) WT mouse, (C) chimeric mouse 10 weeks after FLT with Nagy GFP+ donor cells, and (D) chimeric mouse 10 weeks after FLT with Nagy GFP+ donor cells, which was treated with liposomal clodronate.
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Figure 4. Immunohistochemistry of lung tissue sections using anti-CD68 and anti-GFP antibodies to identify all macrophages (CD68) and donor-derived (GFP+) cells following FLT. WT controls stained for CD68 (A) and GFP (B) demonstrate CD68+ cells but no GFP+ cells, whereas Nagy GFP transgenic mice showed equal numbers of CD68+ (C) and GFP+ (D) cells. In chimeric mice 10 weeks after FLT with GFP+ donor cells, immunostaining for CD68 (E) and GFP (F) shows similar numbers of CD68+ cells compared with untreated controls but fewer GFP+ than CD68+ macrophages. In chimeric mice treated with liposomal clodronate 6 weeks after FLT, the number of CD68+ cells (G) was unchanged, but GFP+ macrophages (H) were increased compared with mice undergoing FLT without subsequent clodronate treatment.
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Figure 5. Quantification of total lung and GFP+ macrophages. Serial lung sections were immunostained to identify the macrophage population (anti-CD68) and donor-derived cells (anti-GFP) in the following groups: Nagy GFP transgenic mice, WT mice, chimeric mice 4 weeks after FLT with GFP+ donor cells (4 Week), chimeric mice 10 weeks after FLT with GFP+ donor cells (10 Week), and chimeric mice 10 weeks after FLT + treatment with liposomal clodronate (10 Week + Clod). Positive cells per field from 10 sequential, nonoverlapping fields per slide were counted. Each bar represents the mean number of positive cells per field ± SEM; n = 8 mice per group. *, P < 0.001, between 4-week GPF+ cells and 10-week GFP+ cells; #, P < 0.001, between GFP+ cells in the 10-week group and GFP+ cells in the 10-week + Clod group.
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Figure 6. Quantification of GFP+ alveolar macrophages in lung lavage from Nagy GFP transgenic mice, WT mice, chimeric mice 10 weeks after FLT with GFP+ donor cells (10 Week), and chimeric mice 10 weeks after FLT + treatment with liposomal clodronate (10 Week + Clod). Cells were collected, labeled with anti-CD11b-PE antibody, and analyzed by FACS to determine the percent of CD11b+ cells that were of GFP+. Each bar represents the mean ± SEM; n = 6 mice per group. *, P < 0.001, between 10-week GFP+ cells and 10-week + clodronate GFP+ cells.
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Figure 7. Quantification of P. aeruginosa colony counts from the lung. WT controls and Nagy GFP FLT chimeras were administered 1 x 106 CFU Pseudomonas. After 24 h, Pseudomonas colony counts from the lung were determined. Each bar represents the mean ± SEM; n = 5 mice per group.
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B in newly recruited alveolar macrophages after IT administration of E. coli LPS (75 µg). Activation of NF-B in macrophages has been shown to be a critical step in the regulation of lung inflammation [15
, 20
]. To detect NF-B activation in alveolar macrophages in vivo, a NF-B transgenic reporter mouse model (HLL) was used in which the NF-B-dependent HIV-LTR promoter drives expression of the Photinus luciferase cDNA [13
, 14
]. FLT chimeras were generated using HLL mice as donors and WT mice as recipients. To maximize alveolar macrophage repopulation, clodronate treatment was performed 6 weeks after transplantation. NF-B activity was detected by bioluminescence imaging, a technique that allows for the detection of luciferase activity in anesthetized, intact mice in vivo. NF-B activity was determined by measuring photon emission over the thorax of mice after a single i.v. dose of 1 mg luciferin. Seven hours after IT LPS administration, quantification of photon counts over the thorax showed a significant increase in NF-B-dependent luciferase activity in the lungs of HLL FLT chimeras compared with baseline (Fig. 8
). As we have previously shown that alveolar macrophages are required for induction of lung NF-B activation following LPS treatment [15
], these data indicate that reconstituted alveolar macrophages are able to respond to E. coli LPS by activating the NF-B pathway.
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Figure 8. Detection of NF-B activity in the lung of HLL FLT chimeras by bioluminescence imaging. Following i.v. injection of luciferin (1 mg), HLL FLT chimeras were imaged at baseline and 7 h after IT administration of E. coli LPS. Quantification of photons (pixels/s/cm2) over the thorax was performed. IT-LPS treatment resulted in a significant increase in NF-B-dependent luciferase activity from the lung compared with baseline (BL). Each bar represents the mean ± SEM; n = 5 mice. *, P < 0.05.
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B activity was determined in lung lavage cells by standard luciferase assays. Seven hours after IT-LPS administration, a significant increase in luciferase activity was detected in lung lavage cells from HLL mice and HLL FLT chimeras [131.5±8.8 and 103.0±24.9 relative light unit (RLU)/µg protein, respectively] compared with untreated HLL controls (26.4±8.0 RLU/µg protein, Fig. 9
). No significant difference was detected between HLL controls and HLL FLT chimeras after IT LPS administration, demonstrating normal induction of NF-B signaling in bone marrow-derived inflammatory cells in the lungs in response to E. coli LPS. Together, experiments using IT administration of LPS show NF-B activation is induced by LPS treatment in donor-derived lung cells in HLL FLT chimeric mice, and the level of LPS-induced NF-B activation in lavage cells is similar in HLL FLT chimeras and (untransplanted) HLL reporter mice.
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Figure 9. Detection of NF-B activity in lung lavage cells by luciferase assay. HLL transgenic reporter mice and HLL FLT chimeras were treated with LPS by IT administration and harvested at 7 h. Untreated WT and HLL mice served as controls. Lung lavage cells were collected, and luciferase activity (RLU) was performed and normalized for total protein. Compared with controls, LPS treatment resulted in a significant increase in luciferase activity in HLL mice and HLL FLT chimeras. Each bar represents the mean ± SEM; n = 5–6 mice per group. *, P < 0.05, compared with WT and HLL mice.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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90% of alveolar macrophages were donor-derived by calculating the ratio of GFP+/CD68+ macrophages by immunohistochemistry in lung tissue sections. These findings were confirmed by FACS analysis of alveolar macrophages obtained by lung lavage.
High-level reconstitution of alveolar macrophages can be obtained by 10 weeks after FLT when recipient macrophages are selectively eliminated. It is interesting that no difference in lung macrophage reconstitution was identified between 10 weeks and 10 months after FLT, implying that there is little advantage to studying mice at time-points later than 10 weeks post-FLT. These studies also indicate that reconstituted macrophages (and neutrophils) following FLT function normally in the clearance of Pseudomonas from the lungs and in the response to E. coli LPS through activation of the NF-B pathway.
Our studies focused primarily on alveolar macrophages, and we did not attempt to discriminate interstitial from alveolar macrophages in the lungs. Although immunodetection of CD68+ and GFP+ macrophages identified the entire population of lung macrophages, the spatial resolution of our immunohistochemistry studies was not sufficient to specifically evaluate the subset of interstitial macrophages, which are less likely to be depleted by IT clodronate treatment [15 ]. The factors that regulate monocyte recruitment, differentiation, and maintenance of the macrophage population in the lungs are not well understood. Murine homolog of monocyte chemoattractant protein-1 (MCP-1; JE)/MCP-1 has been shown to increase alveolar monocyte accumulation after IT instillation, and inflammatory stimuli, such as LPS, increase JE/MCP-1 production and monocyte recruitment [21 ]. It is interesting that following clodronate depletion of lung macrophages, we observed a substantial increase in lung lavage MCP-1 levels induced by LPS treatment, and other cytokines measured were decreased by macrophage depletion [15 ]. This finding implies that macrophages in the lungs may inhibit monocyte recruitment signals, particularly in the presence of inflammatory stimuli. However, further investigations are required to fully define the mechanisms of lung monocyte/macrophage trafficking.
Prior studies have not adequately clarified the kinetics of alveolar macrophage repopulation following bone marrow transplantation or FLT. In the literature, the reported rate of repopulation varies greatly. For example, Ono et al. [22 ] investigated the migration of donor cells from GFP transgenic mice into several tissues in bone marrow chimeras. GFP+ cells were detected in lung sections by fluorescence microscopy and verified as macrophages by labeling with CD11b antibody. This study found that donor cells were present by 7 days, suggesting rapid repopulation. In a separate study, bone marrow chimeras were generated using ROSA 26 mice to track ß-galactosidase-positive cells into various organs [23 ]. In these experiments, donor cells were not observed in the lung until after 1 month. Only 61% of macrophages were of donor origin 1 year after transplant. These data suggest significantly slower kinetics for repopulation of alveolar macrophages and imply that achieving complete repopulation is not possible. The efficiency of bone marrow engraftment and subsequent alveolar macrophage repopulation is likely dependent on several factors, including the method of recipient marrow depletion, the number of donor cells delivered, and the effects of the conditioning treatment on turnover of resident alveolar macrophages. We used a protocol delivering a split radiation dose designed to achieve complete marrow depletion and minimized damage to structural cell components. Others have used different methodologies. For example, Ono et al. [22 ] depleted recipient marrow by intraperitoneal administration of 5-fluorouracil, and Kennedy and Abkowitz [23 ] subjected mice to a single dose of radiation (1050 cGy) from a dual cesium source.
In addition to alveolar macrophages, other bone marrow-derived immune cells, such as neutrophils and eosinophils, are reconstituted with cells of donor genotype following FLT; however, these cells are not present in the lungs in substantial numbers in the absence of an inflammatory stimulus. As alveolar macrophages serve a sentinel function to initiate innate immune responses in the lungs, it is possible to study their function using a bone marrow chimera strategy. We have demonstrated that alveolar macrophage repopulation can be enhanced by elimination of resident alveolar macrophages using liposomal clodronate, and we have defined the extent of alveolar macrophage reconstitution using this methodology. This method will allow efficient replacement of alveolar macrophages with cells of altered genotype to study the role of specific molecules and signal transduction components in alveolar macrophages in vivo.
Received December 22, 2003; revised October 10, 2004; accepted October 12, 2004.
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B-deficient fetal liver cells Immunity 6,765-772[CrossRef][Medline]B activation in a transgenic mouse model of systemic inflammation Am. J. Respir. Crit. Care Med. 162,1095-1101B activation in response to endotoxin Am. J. Respir. Cell Mol. Biol. 26,572-578B activation after intratracheal endotoxin: correlation with cytokine gene expression and neutrophilic alveolitis Am. J. Physiol. 277,L823-L830This article has been cited by other articles:
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