Abstract
Rationale: During acute lung injury (ALI) the macrophage pool expands markedly as inflammatory monocytes migrate from the circulation to the airspaces. As inflammation resolves, macrophage numbers return to preinjury levels and normal tissue structure and function are restored.
Objectives: To determine the fate of resident and recruited macrophages during the resolution of ALI in mice and to elucidate the mechanisms responsible for macrophage removal.
Methods: ALI was induced in mice using influenza A (H1N1; PR8) infection and LPS instillation. Dye labeling techniques, bone marrow transplantation, and surface immunophenotyping were used to distinguish resident and recruited macrophages during inflammation and to study the role of Fas in determining macrophage fate during resolving ALI.
Measurements and Main Results: During acute and resolving lung injury from influenza A and LPS, a high proportion of the original resident alveolar macrophages persisted. In contrast, recruited macrophages exhibited robust accumulation in early inflammation, followed by a progressive decline in their number. This decline was mediated by apoptosis with local phagocytic clearance. Recruited macrophages expressed high levels of the death receptor Fas and were rapidly depleted from the airspaces by Fas-activating antibodies. In contrast, macrophage depletion was inhibited in mice treated with Fas-blocking antibodies and in chimeras with Fas-deficient bone marrow. Caspase-8 inhibition prevented macrophage apoptosis and delayed the resolution of ALI.
Conclusions: These findings indicate that Fas-induced apoptosis of recruited macrophages is essential for complete resolution of ALI.
During acute lung injury, the alveolar macrophage pool in the lungs expands as monocytes are recruited from the circulation to areas of injury. As inflammation resolves, the macrophage pool contracts to baseline levels. The fate of resident and recruited macrophages during the resolution of inflammation is unknown.
Macrophages that are recruited to the lungs during the course of acute lung injury undergo apoptosis as inflammation resolves. Activation of Fas on recruited macrophages drives their death.
Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome, are characterized by bilateral pulmonary infiltrates of noncardiac origin and refractory hypoxemia (1). Despite recent advances, disease mortality remains unacceptably high (2, 3). Moreover, most survivors have persistent pulmonary dysfunction resulting from fibrosis (4–6). Numerous studies have focused on the mechanisms that initiate lung injury; however, few have explored the processes that lead to the resolution of inflammation. In this regard, we hypothesize that alveolar macrophages play an important role.
There is marked expansion of the macrophage pool in the lungs during ALI that occurs as early as 36 hours after the onset of injury (7, 8). In patients with nonresolving lung injury, the macrophage pool remains expanded for up to 28 days (7, 8). Persistence of macrophages at sites of injury is a hallmark of chronic inflammation that is associated with tissue destruction and the development of fibrosis (9, 10). Conversely, elimination of macrophages from sites of chronic injury leads to rapid resolution (11, 12). The primary goal of our study was to determine the fate of inflammatory macrophages in self-limited models of ALI and to elucidate the mechanisms that drive clearance of the macrophages.
Removal of inflammatory macrophages has been studied in model systems outside of the lungs. In the peritoneum, macrophages migrate to the draining lymph nodes during resolving injury (13, 14). Similarly, efflux of mononuclear phagocytes to the lymphatics is essential for resolution of inflammation in the kidneys and vasculature (15, 16). In contrast, elimination of inflammatory macrophages through apoptosis is required for resolution of injury in the skin, muscles, and nervous system (17, 18). Heretofore, the mechanisms that drive contraction of the inflammatory macrophage pool in resolving lung injury have not been fully determined. Moreover, it is unknown whether the macrophages that are present in the airspaces before the onset of injury (i.e., resident alveolar macrophages) remain in the lungs after inflammation resolves or if they are replaced by recruited macrophages.
In this study we used bone marrow chimeras and dye labeling techniques to track the kinetics of resident and recruited alveolar macrophages under homeostatic conditions and after LPS-induced lung injury. Under noninflammatory conditions resident alveolar macrophages were shown to have a prolonged lifespan with minimal replacement by bone marrow–derived cells. During acute and resolving inflammation, a high proportion of the original resident alveolar macrophages persisted. In contrast, recruited macrophages exhibited robust accumulation in early inflammation followed by a progressive decline in their number. Fas-induced apoptosis of these macrophages with local phagocytic removal was shown to be responsible for this accelerated turnover and efficient resolution of inflammation. These findings were confirmed in mice with ALI caused by influenza A (H1N1) infection.
Detailed methods are provided in the online supplement.
This study was approved by and performed in accordance with the ethical standards of the Institutional Animal Care and Use Committee at National Jewish Health, Denver, Colorado. All mice were obtained from the Jackson Laboratory (Bar Harbor, ME).
Isolation of bone marrow was performed as described (19). Recipient mice were anesthetized with Avertin (2–2-2 tribromoethanol) and then positioned between lead strips 1-cm thick by 2-cm wide to protect the lungs from radiation. Radiation was provided as 900 cGy of total body radiation. Six hours after irradiation, recipient mice received 5 × 107 bone marrow cells. Experiments were performed at least 6 weeks after bone marrow transplantation.
LPS (Escherichia coli O55:B5 from List Biological Laboratories, Campbell, CA) was instilled directly into the tracheas of mice lightly sedated with isoflurane using a modified feeding needle. Mouse-adapted influenza A/Puerto Rico/8/34 (H1N1; PR8) was a kind gift from Dr. Kevan Hartshorn (Boston, MA) (20). Fluorescent focus units of 50 × 103 were given intranasally in 50-μl sterile phosphate-buffered saline (PBS).
PKH26-Phagocytic Cell Linker (PCL; Sigma-Aldrich, St. Louis, MO) was diluted in Diluent B (20 μM) and instilled directly into the lungs of mice. LPS was administered at least 24 hours after PKH to ensure selective labeling of resident alveolar macrophages.
Bronchoalveolar lavage (BAL) was performed as previously described (19). Leukocytes were quantified using a hemacytometer. Cell differentials were determined using Wright-Giemsa–stained cytopsin specimens. Cells from BAL were washed twice and then resuspended in PBS containing 2% paraformaldehyde for flow cytometry experiments. Albumin was measured on cell-free supernatant from the first milliliter of BAL fluid using ELISA (Bethyl Laboratories, Montgomery, TX).
Fas-activating experiments were performed using the Jo2 anti-CD95 monoclonal antibody. Experimental mice were treated with LPS (20 μg). Six days later, the Fas-activating antibody was administered intratracheally (20 μg in 50 μl PBS). An equivalent dose of hamster IgG2 (clone Ha4/8) was administered as a control. BAL was performed 24 hours after Fas-activation. Fas-blocking experiments were performed using hamster anti-mouse CD178 monoclonal antibody (clone MFL3) or purified hamster IgG1 (clone A19–3) as a control. The Fas-blocking antibody (50 μg in 50 μl PBS) was administered intratracheally on Days 4 and 7 after LPS. BAL was performed on LPS Day 10.
The selective inhibitor of caspase-8 (Z-IETD-FMK, BD Biosciences, Franklin Lakes, NJ) and its control compound Z-FA-FMK were administered intraperitoneally (0.1 μM in 100 μl PBS) starting on Day 4 after LPS and then daily for a total of 6 days. BAL was performed on LPS Day 10.
Flow cytometry was performed on paraformaldehyde-fixed cells as described (19). FcγR was blocked using anti-CD16/CD32 for 20 minutes. Cells were incubated with 1 μg of primary antibody on ice for 30 minutes, washed twice, and then taken to flow cytometry. Staining with annexin V and propidium iodide was performed on unfixed cells using the Vybrant apoptosis kit (Invitrogen, Carlsbad, CA). Flow cytometry was performed using a FACScan cytometer (Becton Dickinson, Franklin Lakes, NJ). Data were collected using Cellquest software (Becton Dickinson) and analyzed with Flowjo software (Tree Star, Ashland, OR). Cell sorting was performed using a Moflo XDP (Dako, Glostrup, Denmark) on unfixed specimens.
Mouse lungs were inflated with low melt agarose and fixed in 4% paraformaldehyde. Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) staining was performed with the Dead End Fluorometric TUNEL System (Promega). Macrophages were identified with Mac-3 (BD Biosciences). Images were acquired with an Axiovert 200M Marianas digital microscopy workstation (Carl Zeiss, Oberkochen, Germany) equipped with 3I Slidebook (Denver, CO) imaging software.
Each slide was evaluated independently by two investigators blinded to the treatment group. Each investigator scored 10 random fields per slide at ×40 magnification. Within each field, points were assigned on a scale from 0–2 for the following criteria: (A) polymorphonuclear leukocyte in the alveolus, (B) polymorphonuclear leukocyte in the interstitium, (C) hyaline membranes, (D) proteinaceous debris in the airways, and (E) alveolar septal thickening. The injury score was calculated according to the following formula: Score = (20 × A + 14 × B + 7 × C + 7 × D + 2 × E) / (number of fields × 100). Injury scores were averaged for the two investigators.
Data are presented as mean ± SEM and include at least three independent experiments. Statistical analysis was performed using two-tailed Student t test for unpaired samples. For multiple comparisons, data were evaluated by analysis of variance with post hoc analysis by the two-tailed Dunnett test.
Central to determining the fate of resident and recruited macrophages during resolving lung injury is establishing the rate of alveolar macrophage turnover during homeostasis. A widely accepted method for measuring turnover of endogenous leukocytes is allogeneic bone marrow transplantation in which the bone marrow of experimental animals is ablated with total body radiation. As an unintended consequence, macrophage function can be altered and turnover can be accelerated (19, 21). To overcome this obstacle we established a bone marrow transplant system in which the lungs of wild-type C57BL/6J mice were protected by lead shields during irradiation (see Figure E1 in the online supplement). Adoptive transfer of bone marrow from Green fluorescent protein (GFP)-expressing donor mice resulted in chimeric animals with bone marrow and peripheral blood of donor origin (Figure E2). Within 21 days of transplantation over 95% of circulating leukocytes expressed GFP. This high level of engraftment was maintained for up to 8 months. Importantly, bone marrow transplantation had no effect on alveolar macrophage numbers (Figure 1A). In addition, macrophages were predominantly GFP−, indicating that they were of pulmonary origin rather than bone marrow–derived (Figures 1B–1D). The bone marrow contribution to alveolar homeostasis remained minimal for up to 8 months after transplantation, such that greater than 70% of alveolar macrophages remained GFP− at Day 240.
Figure 1. Resident alveolar macrophage turnover at baseline. (A–D) Bronchoalveolar lavage (BAL) was performed on lung-shielded Green fluorescent protein (GFP) chimeras after bone marrow transplantation (n = 8 per time point). (A) Total alveolar macrophage counts in BAL. The dashed line represents the mean for all samples. (B) Alveolar macrophage GFP expression. Macrophages were identified by their forward-scatter and side-scatter properties (R1) and the macrophage marker, F4/80 (R2). GFP was used to discriminate alveolar macrophages (AM) of pulmonary origin (R3) from macrophages of bone marrow origin (R4). Representative flow cytometry plots from BAL performed 30 days after transplantation are shown. (C and D) The percentage and total number of GFP-negative alveolar macrophages (i.e., recipient origin) are shown (n = 6–8 per time point). The dashed line represents the mean for all samples. (E) Naive mice were treated with intratracheal PKH26-PCL dye. BAL was performed at 1, 15, or 30 days later. PKH fluorescence intensity was assessed on F4/80+ macrophages. (F) Peritoneal lavage from abdomen-shielded GFP chimeras (n = 4 per time point). F4/80+ peritoneal macrophages (PM) were assessed for GFP expression.
[More] [Minimize]To confirm the findings of the bone marrow transplant model and to exclude local proliferation as a mechanism for maintenance of the alveolar macrophage pool the phagocyte-specific fluorescent dye PKH26-PCL was instilled directly into the lungs of wild-type mice. Alveolar macrophage levels were unchanged after PKH treatment. More importantly, the fluorescent intensity of PKH26 labeling remained stable for up to 30 days (Figure 1E). Taken as a whole, the results of our experimental models suggest that the turnover rate of unstimulated alveolar macrophages is exceptionally low with a half-life that exceeds 12 months.
Alveolar macrophages exist in a unique extraepithelial environment that communicates directly with the atmosphere, contains high partial pressures of oxygen, and contains high levels of surfactant lipids. To compare the turnover rate of alveolar macrophages with macrophages from other tissue compartments, lead shielding of the abdomen was used to protect peritoneal macrophages during radiation and bone marrow transplantation. Peritoneal lavage performed on GFP chimeras 3, 5, or 7 weeks after transplantation demonstrated rapid replacement of host-derived (i.e., GFP−) peritoneal macrophages by macrophages of bone marrow origin (Figure 1F). The corresponding half-life of peritoneal macrophages was 15 days, further highlighting the unique turnover kinetic of the alveolar macrophage.
We next sought to determine if the prolonged lifespan of resident alveolar macrophages extended into the context of inflammation. GFP bone marrow chimeras were created with the lungs shielded during radiation. Six weeks later the animals were treated with high-dose LPS (200 μg) via direct intratracheal instillation. This resulted in a robust influx of neutrophils into the air spaces that peaked at 72 hours and resolved within 12 days (Figure 2A). Macrophage counts peaked in parallel but took over 30 days to return to baseline. Analysis with flow cytometry revealed that cells recruited from the bone marrow and circulation (i.e., GFP+ cells) were solely responsible for the increase in macrophage numbers (Figure 2B). In sharp contrast, resident alveolar macrophage counts declined by 40% in the first 72 hours after LPS (P < 0.05) but then remained remarkably constant for the next 8 months.
Figure 2. Macrophage kinetics in the lungs during LPS-induced injury. (A and B) Lung-shielded bone marrow chimeras were treated with high-dose (200 μg) intratracheal LPS (n = 12 per time point). (A) Leukocyte counts from bronchoalveolar lavage (BAL). (B) Resident (Green fluorescent protein negative [GFP−]) and recruited (GFP+) macrophages discriminated on the basis of GFP expression. (C and D) BAL from mice treated with PKH26-PCL followed by LPS (200 μg) (n = 6 per group). (C) PKH26 fluorescent intensity on F4/80+ alveolar macrophages (AM). Representative histograms are shown. (D) F4/80+ PKHhigh and F4/80+ PKHlow cell populations sorted from BAL. Wright-Giemsa–stained cytospin samples from LPS Day 6 are shown (×60). (E) PKH labeling on F4/80+ macrophages lavaged from GFP bone marrow chimeras treated with PKH26-PCL followed by LPS. The corresponding histogram displays PKH fluorescence for resident (F4/80+ GFP−) and recruited (F4/80+ GFP+) AM. (F) Resident and recruited AM counts in BAL from wild-type mice treated with PKH and LPS (200 μg). Resident macrophages were defined as F4/80+ PKHhigh cells. Recruited macrophages were defined as F4/80+ PKHlow cells (n = 6 per group). * P < 0.05 versus Day 0.
[More] [Minimize]To confirm these findings, PKH26-PCL was used to distinguish resident and recruited macrophages during LPS-induced inflammation. Resident alveolar macrophages were selectively labeled by instilling PKH26-PCL directly into the lungs. Intratracheal LPS (200 μg) was administered 48 hours later. BAL specimens from LPS-treated mice contained a discrete population of F4/80+ PKHhigh cells with the same fluorescent intensity as resident alveolar macrophages from noninflamed mice. In addition, a large peak of F4/80+ PKHlow cells was present during early inflammation that resolved over time (Figure 2C). We hypothesized that the PKHhigh population represented resident alveolar macrophages, whereas recruited mononuclear phagocytes comprised the PKHlow population. To test this hypothesis the PKH populations were sorted using fluorescence-activated cell sorter and their morphologies were examined by light microscopy. F4/80+ PKHhigh cells exhibited a smooth nuclear contour and morphology consistent with mature macrophages. F4/80+ PKHlow cells had cleaved nuclei with irregular contours and morphology resembling less mature macrophages (Figure 2D). As a final step to validate the PKH labeling technique, lung-shielded GFP chimeras were treated with PKH followed 48 hours later by LPS. Analysis of BAL specimens with flow cytometry demonstrated that resident alveolar macrophages (i.e., F4/80+ GFP− cells) stained intensely with PKH (Figure 2E). In comparison, recruited macrophages (i.e., F4/80+ GFP+ cells) were PKHlow. Using the PKH model, resident and recruited macrophage populations were quantified in wild-type mice treated with LPS (Figure 2F). In concordance with the bone marrow transplantation model, resident alveolar macrophage levels remained stable throughout the course of inflammation.
To shorten the inflammatory time course for subsequent experiments, kinetic studies were repeated with a lower dose of LPS (20 μg) using GFP bone marrow chimeras. Neutrophil influx peaked 3 days after LPS administration and resolved by Day 6 (Figure 3A). Macrophage counts peaked at Day 6 and returned to baseline by Day 12. Recruited mononuclear phagocytes (F4/80+GFP− cells) accounted for the bulk of the macrophage increase (Figure 3B). Twelve days after the administration of LPS, recruited macrophages were still present in the BAL but their numbers were reduced by 75% from their peak. In comparison, resident alveolar macrophage levels remained constant throughout the time course. Notably, there was no significant change in resident alveolar macrophage numbers during early inflammation, unlike the high-dose model in which a 40% decline was observed in the immediate post-LPS period. Lung injury was further quantified by calculating histologic lung injury scores (Figure 3C) and by measuring albumin concentrations as an indicator of alveolar capillary permeability (Figure 3D). The lower-dose (20 μg) LPS model was used for all subsequent experiments.
Figure 3. Lung injury after low-dose LPS (20 μg) in lung-shielded bone marrow chimeras (A and B) and wild-type mice (C and D). (A) Leukocyte counts from BAL. (B) Resident (F4/80+ Green fluorescent protein [GFP]−) and recruited (F4/80+ GFP+) macrophages discriminated on the basis of GFP expression (n = 8 per group). (C) Histologic lung injury scores from tissue sections stained with hematoxylin and eosin. (D) Albumin concentrations in BAL fluid (n = 6 per group). * P < 0.05 versus Day 0.
[More] [Minimize]A limitation of both the bone marrow transplant model and the PKH labeling technique was the potential to alter macrophage programming by the interventions used for their identification. In the former, small doses of radiation pass through the shields and in the latter a foreign substance is introduced directly into the lungs. We therefore asked whether surface immunophenotyping could be used to distinguish resident and recruited macrophages during inflammation. In this regard, unstimulated alveolar macrophages exhibit a unique pattern of β2-integrin expression that includes high levels of CD11c, and compared with other tissue macrophages relatively low levels of CD11b (22). CD11b expression is up-regulated on peripheral blood monocytes during inflammation and is required for their successful migration to inflamed tissues (23). This line of evidence suggested that the β2-integrin expression profile on resident alveolar macrophages would be CD11chigh and CD11blow, whereas freshly recruited macrophages would be CD11clow and CD11bhigh. To test this hypothesis, BAL was performed on lung-shielded GFP chimeras treated with intratracheal LPS. F4/80+ macrophages were analyzed for β2-integrin expression both directly and after gating on GFP− and GFP+ populations (Figure 4A). Unstimulated alveolar macrophages expressed high levels of CD11c and relatively low levels of CD11b. After LPS administration, resident alveolar macrophages transiently up-regulated their expression of CD11b but maintained constant levels of CD11c. By Day 9, β2-integrin expression on resident macrophages reverted to that of unstimulated macrophages (i.e., CD11chigh/CD11blow). In comparison, recruited macrophages expressed high levels of CD11b and low levels of CD11c at early time points. As inflammation resolved, they began to gain CD11c expression while maintaining high levels of CD11b. At the resolution of inflammation (Day 12) a mixed population of recruited macrophages existed, some of which were CD11chigh. These data demonstrated qualitatively that for the first 9 days of the inflammatory time course, surface immunophenotyping with CD11b and CD11c tracked closely with GFP expression. To quantify these results, the number of resident alveolar macrophages in each BAL sample was independently determined based on GFP expression and with β2-integrin expression. When compared with each other, these values showed excellent correlation (r2 = 0.919; Figure E3). Recruited macrophages were similarly quantified and also demonstrated excellent correlation (r2 = 0.918).
Figure 4. Differential expression of CD11b and CD11c distinguishes resident and recruited alveolar macrophages. (A) Dot plots representing CD11b and CD11c staining on macrophage subpopulations lavaged from lung-shielded Green fluorescent protein (GFP) chimeras treated with LPS (20 μg). Macrophages were identified with F4/80 mAb (R1). Resident alveolar macrophages were defined as GFP− cells (R2), whereas recruited macrophages were defined as GFP+ (R3). Dot plots representing CD11b and CD11c staining on macrophage subpopulations shown for each time point are representative of three independent bone marrow transplant experiments. (B and C) Resident (F4/80+ CD11chigh CD11blow) and recruited (F4/80+ CD11clow CD11bhigh) macrophages from wild-type mice treated with 20 μg LPS (n = 8 mice per time point). * P < 0.05 versus Day 0. FSC = Forward Scatter.
[More] [Minimize]Because surface immunophenotyping provided a robust tool to distinguish resident and recruited macrophages without the potential to alter macrophage phenotype, it was used to verify macrophage kinetics in wild-type mice treated with low-dose LPS. In concordance with findings from bone marrow transplant experiments and PKH labeling studies, resident alveolar macrophage levels remained stable during inflammation (Figure 4B). The kinetics of recruited macrophages further confirmed findings from the other models. As shown in Figure 4C, high numbers of recruited macrophages were present in the airspaces by Day 3. As inflammation resolved, there was steady contraction of the recruited macrophage pool.
The fate of recruited macrophages during resolving lung injury is unknown. We hypothesized that apoptosis followed by in situ phagocytosis would be the major mechanism driving contraction of the recruited macrophage pool. To distinguish resident and recruited macrophage populations, bone marrow transplantation with lung shielding was performed. As with previous experiments, wild-type C57Bl/6 mice (which express the alloantigen CD45.2) were used as recipient mice. B6.SJL-Ptprca (which express the alloantigen CD45.1) mice were selected as donors instead of GFP-expressing mice because the assays used to quantify apoptosis required fluorescence in the same wavelength as GFP. LPS was administered to bone marrow chimeras 6 weeks after transplantation and BAL was performed 6, 9, or 12 days later. Flow cytometry was used to assess annexin V and propidium iodide staining on resident (CD45.2) and recruited (CD45.1) macrophages. As shown in Figure 5A, the percentage of resident alveolar macrophages that were annexin positive was low at baseline and during resolving inflammation. In comparison, a significant percentage of recruited macrophages became annexin positive. Recruited macrophages were also positive for propidium iodide on LPS Day 12 (Figure 5B).
Figure 5. Recruited macrophages undergo apoptosis during resolving inflammation. (A and B) Annexin V and propidium iodide staining on resident and recruited alveolar macrophages lavaged from CD45.1 bone marrow chimeras. Resident macrophages were F4/80+ CD45.1− and recruited macrophages were F4/80+ CD45.1+. n = 12 for all groups except Day 9 where n = 8. * P < 0.05 versus Day 0. (C) Activated caspase-3 staining on resident (F4/80+ CD11chigh CD11blow) and recruited (F4/80+ CD11clow CD11bhigh) macrophages. n = 10 per group; * P < 0.05 versus Day 0. (D) Macrophage cell death in paraffin-embedded tissue sections using terminal deoxynucleotidyltransferase-mediated dUDP nick-end labeling (TUNEL; red). Macrophages were identified using Mac-3 Ab (green) and were distinguished from autofluorescent erythrocytes based on cell morphology and the presence of nuclear staining with DAPI (blue). Sections incubated with DNase-1 provided a positive control for TUNEL. Negative controls for TUNEL were processed without the enzyme terminal deoxynucleotidyl transferase. Apoptotic macrophages displayed intense TUNEL staining that colocalized with DAPI nuclear staining (large arrow). Macrophages containing ingested apoptotic cells were also present (short arrowhead). (E) Cytospin samples from LPS-treated chimeras were used to identify macrophages that had ingested apoptotic cells. Representative images are shown (×60). Arrows indicate ingested apoptotic cells.
[More] [Minimize]To confirm that annexin staining on recruited macrophages represented apoptosis, activated caspase-3 was assessed using flow cytometry. As shown in Figure 5C, a greater percentage of recruited macrophages were positively stained. As a final step to confirm macrophage cell death, tissue sections from LPS-treated mice were analyzed with TUNEL and mac-3 staining. Numerous TUNEL-positive macrophages were present during resolving inflammation (Figure 5D). No apoptotic cells were detectable in tissue sections from uninjured animals. Additional images and control sections are shown in Figure E4.
To determine if dying macrophages were cleared locally, cytospin preparations from LPS-treated mice were examined for evidence of macrophage phagocytosis. During time points at which macrophages were the predominant cells (>95%) that could be lavaged from the airspaces (i.e., 6 and 9 d after LPS) ingested apoptotic bodies were visible in macrophage phagosomes (Figure 5E). Taken as a whole, these findings suggested that during resolving lung injury recruited macrophages underwent in situ cell death and were rapidly cleared by neighboring macrophages. In separate experiments migration to the lymph nodes was ruled out as an additional mechanism for contraction of the macrophage pool (Figure E5).
The death receptor Fas is a critical regulator of monocyte lifespan in the bloodstream. Because recruited macrophages are derived from circulating monocytes, experiments were performed to determine if they expressed Fas during resolving inflammation. As shown in Figure 6A, recruited macrophages (defined as F4/80+ CD11clow CD11bhigh cells) expressed high levels of Fas during resolving lung injury. In comparison, low levels of Fas were present on resident alveolar macrophages (F4/80+ CD11chigh CD11blow cells) at baseline and during inflammation. When Fas was quantified using mean fluorescent intensity, these differences were highly significant (Figure 6B). In separate experiments, these findings were confirmed using GFP bone marrow chimeras.
Figure 6. Recruited mononuclear phagocytes express high levels of Fas. (A) Fas expression on recruited (F4/80+ CD11clow CD11bhigh) and resident (F4/80+ CD11chigh CD11blow) macrophages from LPS-treated mice. (B) Fas mean fluorescent intensity (MFI) for resident and recruited macrophages. Δ MFI = MFI Fas – MFI Isotype. n = 8 mice per group. ** P < 0.001 versus resident Δ MFI.
[More] [Minimize]To assess the functional significance of Fas expression on recruited macrophages, a Fas-activating antibody was administered to LPS-injured mice during resolving inflammation. The antibody reduced macrophage levels significantly compared with the isotype control (Figure 7A). Depletion of recruited macrophages was responsible for the reduction. The percentage and overall number of CD11clow CD11bhigh recruited mononuclear phagocytes was reduced in mice treated with the anti-Fas antibody, whereas resident macrophage (CD11clow CD11bhigh) numbers were unaffected (Figures 7B and 7C). These results suggest that Fas expressed on recruited mononuclear phagocytes was functional.
Figure 7. Administration of a Fas-activating antibody depletes recruited mononuclear phagocytes from LPS-injured lungs. LPS-injured mice were treated with an agonistic Fas antibody (or isotype control) during resolving inflammation. (A) BAL macrophage counts 24 hours after Ab administration. (B) CD11c and CD11b expression on F4/80+ cells. (C) Recruited (F4/80+ CD11clow/CD11bhigh) and resident (F4/80+ CD11chigh/CD11blow) macrophages were quantified for Fas antibody–treated mice (white bars) and mice treated with the isotype control antibody (black bars). n = 12 mice per group. * P < 0.05 for Fas versus isotype.
[More] [Minimize]To demonstrate that activation of Fas was required for the resolution of macrophage inflammation, experiments were performed to block Fas. As shown in Figure 8A, the administration of a Fas-blocking antibody to LPS-injured mice prevented contraction of the inflammatory macrophage pool. In comparison, macrophage levels in mice treated with an isotype control antibody were reduced by over 50%.
Figure 8. Fas is required for contraction of the inflammatory macrophage pool. (A) Wild-type mice were treated with LPS (20 μg) followed a Fas-blocking antibody (administered on LPS Days 4 and 7) or isotype control. Macrophage counts from lavage. n = 10 mice per group; * P < 0.05 versus isotype control. (B–D) Bone marrow chimeras were created by adoptively transferring bone marrow from Fas knockout (Fas -> Green Fluorescent Protein [GFP]) or wild-type mice (WT -> GFP) into GFP-expressing recipients that were irradiated with lead shields protecting the lungs. (B) Total macrophages in lavage on Day 4 (n = 4 per group) and Day 10 (n = 8 for wild-type chimeras, n = 11 for Fas chimeras). (C) Recruited (F4/80+ GFP−) macrophages in lavage on Day 10. (D) Histologic lung injury scores for LPS-treated chimeras. * P < 0.05, ** P < 0.01 for Fas knockout versus wild-type chimeras.
[More] [Minimize]To confirm that Fas was necessary for depletion of the recruited macrophage pool, bone marrow from Fas-deficient mice (Faslpr/Faslpr) was transplanted into GFP-expressing mice that had been irradiated with lead shields protecting the lungs. Resultant chimeras had resident (i.e., GFP+) alveolar macrophages that expressed Fas and recruited (i.e., GFP−) macrophages that were Fas-deficient. GFP chimeras with wild-type bone marrow served as controls. Macrophage counts were similar between wild-type and Fas-deficient chimeras during acute inflammation but two-fold higher in Fas-deficient chimeras during resolving injury (Figure 8B). Recruited macrophages accounted for this difference (Figure 8C). Importantly, Fas-deficient chimeras had delayed resolution of lung injury scores (Figure 8D) suggesting that macrophage apoptosis is required for the resolution of lung injury.
Ligation of Fas leads to receptor cross-linking, formation of the death-induced signaling complex, and proteolytic cleavage of procaspase-8 to form activated caspase-8 (24). We therefore hypothesized that pharmacologic inhibition of caspase-8 during resolving inflammation would prevent alveolar macrophage apoptosis and delay the resolution of lung injury. To test this hypothesis the caspase-8 selective inhibitor, Z-IETD-FMK, was administered to LPS-injured mice at the peak of macrophage accumulation and then daily for a total of 6 days. The compound Z-FA-FMK was used as a negative control. Caspase-8 inhibition fully abrogated contraction of the macrophage pool (Figure 9A) and delayed resolution of lung injury as assessed by histologic lung injury scores and BAL albumin concentrations (Figures 9B and 9C).
Figure 9. Caspase-8 inhibition prevents contraction of the macrophage pool. Wild-type mice were treated with LPS followed 4 days later by a selective inhibitor of caspase-8 (Z-IETD-FMK, 0.1 μM), phosphate-buffered saline, or control (Z-FA-FMK 0.1 μM) daily for 6 days. Gray bars represent treatment-naive mice. (A) Macrophage counts in bronchoalveolar lavage (BAL) (n = 10 per group). (B) Lung injury scores from tissue sections (n = 5 per group). (C) Albumin concentrations in BAL (n = 5 per group). * P < 0.05 versus control; # P = 0.09 versus control.
[More] [Minimize]Community-acquired pneumonia represents the most common cause of ALI in humans (25). Most recently, influenza A (specifically the H1N1 strain) has received significant attention as a causative agent. To determine if macrophage responses and cell fate in influenza-induced ALI parallel those of LPS-induced lung injury, we infected mice with mouse-adapted H1N1; PR8 virus (20). Infection induced robust accumulation of neutrophils, macrophages, and lymphocytes in the airways that peaked 7 days after inoculation (Figure 10A). Lung injury was further evaluated with histology and BAL albumin concentrations (Figures 10B and 10C). In concordance with the LPS-induced lung injury model, resident alveolar macrophage numbers (i.e., F4/80+ CD11chigh CD11blow cells) remained constant throughout the course of injury, whereas recruited macrophages (F4/80+ CD11clow CD11bhigh cells) were evident in the BAL as early as 4 days after infection and peaked at Day 7 (Figure 10D). The use of β2-integrins to distinguish resident and recruited macrophages was validated in separate experiments by infecting GFP-expressing bone marrow chimeras with H1N1 (data not shown). Alveolar macrophage cell death was quantified in histologic tissue sections using TUNEL staining (Figure 10E). Taken as a whole, the findings from the H1N1-induced lung injury model correspond closely with findings from the LPS injury model and suggest that resident alveolar macrophages persist throughout the full course of inflammation. In comparison, recruited macrophages exhibit robust accumulation in the airspaces during early inflammation and then undergo programmed cell death during resolving inflammation.
Figure 10. Lung injury after influenza A infection (H1N1; PR8). Lung injury was assessed using (A) bronchoalveolar lavage (BAL) cell counts, (B) BAL albumin concentrations, and (C) lung histology. (D) Resident (F4/80+ CD11chigh CD11blow) and recruited (F4/80+ CD11clow CD11bhigh) macrophage levels in BAL fluid. (E) Macrophage cell death quantified in tissue sections using terminal deoxynucleotidyltransferase-mediated dUDP nick-end labeling (TUNEL). n = 5 mice per group * P < 0.05 versus Day 0.
[More] [Minimize]During ALI there is marked expansion of the macrophage pool driven by migration of inflammatory monocytes from the circulation. Restoration of alveolar function and resolution of inflammation are accompanied by return of macrophage numbers to their preinjury levels. The goal of our study was to determine the fate of resident and recruited macrophage populations during resolving lung injury. Herein we demonstrate that resident alveolar macrophages exhibit a slow turnover kinetic in the absence of inflammation and that a significant number survive through the inflammatory cycle. In contrast, during resolving lung injury recruited macrophages undergo Fas-mediated cell death, such that only a small fraction remains after the resolution of inflammation.
The slow turnover kinetic of resident alveolar macrophages under noninflammatory conditions is an important finding of our study. Other investigators have observed this result using radiation-limited bone marrow transplantation techniques that are complementary to ours (26–28). However, an inherent problem with bone marrow transplant techniques is that local replication cannot be excluded as a source for macrophage replacement. Indeed, several groups have suggested that this may occur during homeostasis (28, 29) or in response to inflammation (30). Because the fluorescent intensity of PKH is halved with each cell division, the stability of high-intensity fluorescent staining in our dye labeling experiments suggests that, at the very least, the rate of resident macrophage replication is low under noninflammatory conditions.
A significant limitation of traditional methods of bone marrow transplantation is the requirement for total body radiation. Excessive radiation can lead to inflammation, organ injury, and fibrosis (31–34). Conversely, inadequate radiation doses may allow host bone marrow cells to survive, leading to failed or incomplete engraftment (33, 35, 36). To strike a balance between these concerns, lead strips were used to shield the thoraxes of recipient mice during radiation. Careful analysis of chimeric animals revealed that over 95% of the leukocytes in the peripheral circulation, spleen, and bone marrow of the femurs were of donor origin. Surprisingly, up to 85% of the leukocytes isolated from the ribs were also of donor origin, even though the entire thorax was covered by lead shields during irradiation. Because our analysis did not distinguish between stem cells and mature leukocytes, it is possible that some of the donor-derived cells originated in hematopoietic compartments outside of the thorax and were in transit at the time the organs were removed. Moreover, the yield of leukocytes from the ribs was significantly lower than the yield from the femur (not shown). Taken together, these findings suggest that the bones of the thorax provide only a small contribution to the circulating pool of leukocytes.
The mechanisms that underlie the low turnover rate of resident alveolar macrophages have only been partly elucidated. In this regard, the unique environment in which the alveolar macrophage resides may be an important determinant because macrophages in other organs including the peritoneum, liver, spleen, and eye exhibit much higher turnover rates (37, 38). Indeed, rapid turnover of peritoneal macrophages occurred in abdomen-shielded chimeras. Similarly, virtually all of the macrophages in thymuses from lung-shielded chimeras were of donor origin by Day 60. Alveolar macrophages are bathed in fluid that contains high levels of surfactant proteins (SP). Binding of SP-A and SP-D to signal inhibitory receptor protein α on the alveolar macrophage surface leads to tyrosine phosphorylation of signal inhibitory receptor protein α and activation of SHP-1 and SHP-2 (39). In turn, activation of SHP-2 leads to upregulation of the prosurvival molecules Akt and ERK (40). This pathway may explain our recent observation that alveolar macrophages from SP-D null mice contain lower levels of ERK than macrophages from wild-type mice and that alveolar macrophage turnover is enhanced in SP-D–deficient mice (unpublished observations). Supporting this concept are studies that demonstrate a protective, antiapoptotic effect of SP-A on type II alveolar epithelial cells (41). High intracellular levels of acid ceramidase may also contribute to the prolonged lifespan of resident alveolar macrophages because this enzyme enables the conversion of ceramide to sphingosine, resulting in sphingosine-dependent activation of ERK and the PI3k downstream effector, Akt (42).
A second major finding of our study is that a significant percentage of resident alveolar macrophages persist through the inflammatory cycle. In both the influenza A and low-dose LPS models of lung injury levels of resident alveolar macrophages in lavage fluid remained constant throughout the course of inflammation. In the high-dose LPS model (200 μg), resident alveolar macrophage numbers dropped by 40% almost immediately after LPS administration but then remained stable for up to 240 days. Maus and colleagues have performed similar evaluations of macrophage kinetics using CD45 bone marrow chimeric mice. In a model of Streptococcus pneumoniae infection they demonstrated no significant change in resident alveolar macrophage concentrations in lavage fluid over the 7-day course of their study (43). They reported comparable findings in a model of endotoxin-induced lung injury in which resident alveolar macrophage levels remained constant for 14 days after a 20-μg dose of LPS (26). This result mirrors ours. Importantly, in their endotoxin model Maus and colleagues continued to follow resident alveolar macrophage levels well after inflammation resolved. They documented a slow and steady decline in resident alveolar macrophage numbers such that 90 days after LPS treatment only 10% of macrophages was of host origin (i.e., resident macrophages). This latter result varies from our data obtained with high-dose LPS in which resident alveolar macrophage levels remained constant for nearly 240 days. One potential explanation for this discrepancy is a difference in bone marrow transplant techniques. Mice in our study received a single radiation dose of 9 cGy but had their lungs protected by lead shields. In comparison, Maus and colleagues used a split-dose conditioning regimen in which mice received total body radiation with doses of 8 and 4 cGy separated by 3 hours. There is strong evidence to suggest that radiation doses as low as 2 cGy can alter macrophage phenotype and promote their turnover, which may explain the discrepancy in results (28, 44, 45). Despite the noted differences in our models and the discrepant results for very late time points, our study and those of Maus and colleagues share a common theme: a high percentage of resident alveolar macrophages survive through the initial phase of inflammation and persist well into the resolution phase.
The static nature of resident alveolar macrophage levels presents a striking contrast to the dynamic character of recruited macrophage kinetics. In both the H1N1 influenza and LPS injury models, mononuclear phagocytes entered the lungs soon after the onset of injury where they accumulated in the air spaces in large numbers. Shortly after reaching their peak, recruited macrophage numbers began steadily to decline. Commensurate with the drop in recruited macrophage numbers was exposure of phosphatidylserine on their cell surfaces as indicated by annexin V staining. Because inflammatory macrophages can expose phosphatidylserine without becoming apoptotic (46), it was important for us to confirm macrophage death using additional assays. This was done using assays for activated caspase-3 and TUNEL. Importantly, macrophages that contained apoptotic cells in their phagosomes were present in both tissue sections and BAL specimens collected during resolving inflammation. Because no neutrophils were present at these time points, the strong implication from these studies is that the engulfed apoptotic cells were effete macrophages. Another group has demonstrated that alveolar macrophages undergo apoptosis after ingesting infectious pathogens, lending further support to the concept of in situ alveolar macrophage cell death (47).
Our data suggest that apoptotic cell death with local phagocytic clearance is a major mechanism that drives contraction of the recruited macrophage pool. Additional possibilities include expectoration via the mucociliary escalator and migration to the draining lymph nodes. The former represents a scenario that is unique to the lungs because the alveoli and conducting airways are extraepithelial and communicate directly with the external environment. Macrophage efflux to the draining lymph nodes represents the main avenue for inflammatory macrophage removal from the peritoneum and kidneys (12–14). In the lungs, small numbers of alveolar macrophages migrate to the bronchopulmonary lymph nodes during the acute response to bacterial infection (48). We tested whether macrophages migrate to the lymphatics during resolving inflammation by labeling inflammatory macrophages with fluorescent PKH26-PCL during early and late inflammatory time points. Importantly, we detected no PKH+ macrophages in the lymph nodes further supporting the notion that resident alveolar macrophages undergo apoptosis in the airspaces.
Our data suggest that Fas plays a major role in determining macrophage cell fate. Recruited macrophages express high levels of Fas on their surface and the administration of a Fas-activating antibody led to their depletion. Caspase-8 activation was not assessed during these experiments, so it is possible that the cytotoxic effect of the Fas-activating antibody occurred independently of Fas activation. Importantly, antibody blockade of Fas and pharmacologic inhibition of caspase-8 prevented depletion of the macrophage pool, lending further support for the role of Fas. Similar results were observed in experiments incorporating Fas-deficient bone marrow chimeras and in adoptive transfer experiments with Fas-deficient and wild-type alveolar macrophages.
Fas ligand (FasL) is the cognate binding partner for Fas. High levels of both the soluble and membrane-bound forms of FasL are present in the lungs during inflammation (49–51); however, it has been suggested that the membrane-bound form is most critical for inducing apoptosis (52). Potential sources of FasL in the airspaces include inflammatory leukocytes and the epithelial cells that form the alveolus (49, 51). In regard to the former, both macrophages and activated lymphocytes can express FasL. In the peritoneum, γδ T cells use FasL to kill inflammatory macrophages that have ingested bacteria (53). In the lungs, most of the lymphocytes are subepithelial in location; however, a modest number are present in the airspaces during resolving inflammation. Moreover, it has recently been shown that T-cell receptor δ knockout mice have sustained expansion of the alveolar macrophage pool after infection with S. pneumoniae (54). In our model of LPS-induced lung injury we were unable to detect soluble FasL in BAL fluid (data not shown). However, we were able to detect significant levels of membrane bound FasL on CD8+ T cells and on both resident and recruited macrophages (data not shown). This latter observation is particularly intriguing because it has been demonstrated in vitro that macrophages that ingest apoptotic cells up-regulate their expression of FasL and induce apoptosis of bystander leukocytes (55). This presents an attractive paradigm because high numbers of apoptotic neutrophils are cleared from the airspaces by inflammatory macrophages immediately before the recruited macrophage pool begins to contract.
Substantial evidence suggests that the Fas-FasL system plays a pathogenic role in driving inflammation and tissue damage during the acute phases of ALI. Direct intratracheal instillation of FasL or Fas-activating antibodies induces ALI in rodents (56, 57). Conversely, lung injury is significantly attenuated in Fas (lpr)- and FasL (gld)-deficient mice (58, 59). Importantly, the deleterious effects of the Fas-FasL system do not involve myeloid cells, but result instead from epithelial cell activation and apoptosis (59, 60). Our study is the first to demonstrate a beneficial role for Fas in the context of ALI. Importantly, our experiments focused on the processes that drive the resolution of lung injury rather than the processes that initiate lung injury.
An important question that arises from our study relates to the potentially disparate roles that resident and recruited macrophages may play during acute and resolving inflammation. In this regard, both resident and recruited macrophages up-regulate their expression of CD40 and major histocompatibility complex class II during early inflammation (data not shown). As inflammation resolves, expression of these molecules returns to baseline. In line with our previous data, we hypothesize that recruited macrophages are essential for removal of apoptotic neutrophils and pathogenic microbes from the airspaces (61). Conversely, we postulate that resident alveolar macrophages are important for initiating repair of the injured alveolus and for continued immune surveillance in the lungs once inflammation has resolved.
A strength of our study is the use of two independent models of ALI. Intratracheal administration of LPS serves as a model for gram-negative sepsis and gram-negative pneumonia, two of the most common causes of ALI in humans (25). Influenza A (specifically the H1N1 strain) has recently garnered significant attention as an important cause of acute respiratory distress syndrome. In both models we observed robust influx of inflammatory cells into airspaces and interstitium that was associated with epithelial–capillary permeability and protein exudation. These abnormalities are consistent with pathologic findings reported with ALI in humans. A common feature of both models was that resident alveolar macrophages persisted through the full course of inflammation in near-constant levels. In contrast, after their initial recruitment, recruited macrophages underwent programmed cell death that was regulated by selective cell surface expression of Fas.
ALI accounts for nearly 200,000 hospitalizations and 75,000 deaths in the United States each year (2). Mortality rates from ALI remain high and survivors often suffer from persistent pulmonary disease (5, 6). To date, the only intervention that improves survival is low-tidal volume ventilation. Animal models have yielded important insights into the mechanisms that initiate lung injury; however, few studies have focused on the processes that drive its resolution. Our study is the first to demonstrate a role for alveolar macrophage apoptosis in the resolution of ALI. Drugs that enhance macrophage sensitivity to apoptosis are currently being developed for the treatment of malignancy and autoimmune diseases (62, 63). We believe that these agents could also provide an attractive therapy for nonresolving ALI.
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Supported by grants from the National Institutes of Health (HL68864, HL81151, and GM61031; P.M.H.) and the American Heart Association (0675040N).
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.201011-1891OC on March 31, 2011
Author disclosures
Authors' contributions: Experimental design: W.J., L.B., A.M., R.O., M.K., C.J., P.H. Performance of experiments: W.J., L.B., A.M., R.O., M.K., C.J. Data analysis and interpretation: W.J., L.B., R.O., M.K., C.J., P.H. Manuscript preparation: W.J., L.B., M.K., P.H.
