Myeloid cells control termination of lung inflammation through the NF-κB pathway

Abstract

Although acute lung inflammation in response to local or systemic infection involves myeloid and nonmyeloid cells, the interplay between different cell types remains poorly defined. Since NF-κB is a key transcription factor for innate immunity, we investigated whether dysregulated NF-κB activation in myeloid cells impacts inflammatory signaling in nonmyeloid cells and generation of neutrophilic lung inflammation in response to systemic endotoxemia. We generated bone marrow chimeras by fetal liver transplantation of cells deficient in IκBα or p50 into lethally irradiated NF-κB reporter transgenic mice. No differences were apparent between bone marrow chimeras in the absence of an inflammatory stimulus; however, following intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS), IκBα- or p50-deficient bone marrow chimeras showed increased NF-κB activation in nonhematopoietic cells, exaggerated neutrophilic inflammation, and higher mortality compared with untransplanted reporter mice and wild-type bone marrow chimeras. Primary bone marrow-derived macrophages (BMDM) from IκBα^−/− or p50^−/− exhibited increased NF-κB activation and macrophage inflammatory protein-2 production after LPS treatment compared with wild-type cells, and coculture of BMDM with lung epithelial (A549) cells resulted in increased NF-κB activation in A549 cells and excess IL-8 production by these epithelial cells. These studies indicate an important role for inhibitory members of the NF-κB family acting specifically within myeloid cells to limit inflammatory responses in the lungs.

the host recognizes and responds to bacterial pathogens by initiating protective inflammatory responses via the innate immune system. Inflammation must be tightly controlled, however, because uncontrolled inflammation can result in lung tissue injury as evident in the acute respiratory distress syndrome (ARDS). The hallmark of ARDS is neutrophilic alveolitis and increased levels of cytokines and chemokines in the airways (5, 22, 40). Immune cells derived from bone marrow (myeloid cells), particularly alveolar macrophages and neutrophils, are key players in innate immunity in the lungs. Macrophages express a variety of pattern recognition receptors, including Toll-like receptor 4 (TLR4), a receptor for gram-negative bacterial lipopolysaccharide (LPS) that is crucial for host innate immunity (26, 29, 32, 42). We and others have shown that macrophages are required for initiation of innate immunity in the lungs in response to LPS and other inflammatory stimuli (8, 21, 25). Depletion of macrophages by treating mice with liposomal clodronate reduces inflammatory responses in the lungs and impairs clearance of gram-negative bacteria (4, 10, 23). Although a key role for macrophages in initiation of inflammatory responses in the lungs has been well established, important functions for these cells in limiting or terminating inflammatory responses are not well defined.

In addition to macrophages, structural cells in the lungs (including epithelial cells) are essential for generating the full innate immune response to local and systemic stimuli. We have recently shown that local and systemic LPS treatment results in prominent activation of the NF-κB pathway in airway epithelial cells (15) and that NF-κB in airway epithelia cells regulates lung inflammation and injury (13, 15, 34). Studies designed to elucidate the interplay between macrophages and lung parenchymal cells are important for developing a more complete understanding of the mechanisms that regulate lung inflammation.

The NF-κB transcription factor pathway plays a crucial role in innate immunity in the lungs and other organs. NF-κB is a ubiquitous transcription factor and functions as a homo- or heterodimer of five members of proteins: c-Rel, RelA (p65), RelB, p50, and p52. The prototypical NF-κB complex is the RelA (p65)/p50 heterodimer, which resides in cytoplasm by forming complexes with inhibitory κB (IκB) in unstimulated conditions (16). IκB includes IκBα, IκBβ, IκBɛ, IκBγ, and the p50 and p52 NF-κB protein precursors p105 and p100 (17). Degradation of IκB results in NF-κB activation. Proinflammatory stimuli such as IL-1β, TNF-α, and LPS induce signal cascades through their cognate receptors, IL-1R, TNFR, and TLR4, to activate the IKK signalsome that phosphorylates IκB. Phosphorylated IκB undergoes ubiquitination and degradation, which results in liberation of NF-κB, translocation of NF-κB to the nucleus, and activation of target genes, including cytokines and chemokines (19). Activation of NF-κB results in upregulation of transcription of inhibitory components IκBα and p105/p50, which in turn aid in turning off NF-κB pathway signaling (28). In addition to inhibitory effects of p105, p50 homodimers (which lack a transcriptional activation domain) can block NF-κB transcriptional activity by translocating the nucleus and competing with other NF-κB dimers for binding to NF-κB motifs (43).

In the present studies, we investigated whether dysregulated NF-κB signaling in myeloid cells impacts NF-κB signaling in other lung cell types and alters development and progression of lung inflammation following systemic endotoxemia. We generated bone marrow chimeras with myeloid cells deficient in IκBα or p50 and tested whether loss of inhibitory feedback on the NF-κB pathway in these cells impacts the duration and intensity of neutrophilic inflammation in the lungs. We also investigated the effects of IκBα or p50 deficiency in macrophages on LPS-induced NF-κB activation and chemokine production by these cells and cocultured epithelial cells. Together, our data point to a critical role for macrophages in limiting the duration and intensity of neutrophilic lung inflammation.

MATERIALS AND METHODS

Animal model.

Adult transgenic mice (male and female) expressing Photinus luciferase cDNA under control of the proximal 5′ HIV-LTR mice on a C57B6/DBA background (HLL) (9) were used for these studies. Mice containing a homozygous deletion of nfkb1 (p50; C57B/6/129 background) were purchased from Jackson Laboratories. Mice deficient for IκBα (C57B/6/129 background) (11, 12) were used. Homozygous IκBα deficiency results in death by 3–7 days of age (3, 20), so these mice are maintained in a heterozygous state. The studies were approved by the Vanderbilt Institutional Animal Care and Utilization Committee.

LPS administration.

Gram-negative Escherichia coli LPS (serotype 055:B5) was obtained from Sigma (St. Louis, MO). A single dose of 3 μg LPS/g body wt was administered by intraperitoneal injection.

Fetal liver transplantation.

Fetal liver transplant (FLT) experiments were performed as previously described (14). Timed matings were set up with donor mice and females checked on consecutive days until vaginal plugs were observed. On embryonic day (E) 14.5, pregnant females were euthanized by CO₂ inhalation, and the uterus was surgically removed. Fetuses were separated using forceps and placed into culture medium (RPMI 1640; Invitrogen, Carlsbad, CA). Embryonic livers were removed with the aid of a dissecting microscope, pooled into an Eppendorf tube containing 1 ml of RPMI, and stored on ice. Single-cell suspensions were prepared by drawing cells through needles of decreasing bore size (18, 23, and 25 gauge). Recipient mice were lethally irradiated using a ¹³⁷Ce gamma source by giving a split dose of 800 rads followed 3 h later by 400 rads. After irradiation, 2 × 10⁶ donor cells were injected intravenously via tail vein.

Liposomal clodronate.

Liposomal encapsulation of clodronate (dichloromethylene diphosphonate) was performed as previously described (39). Briefly, a mixture of 8 mg of cholesterol (Sigma) and 86 mg of egg phosphatidylcholine (DOPC; Avanti, Alabaster, AL) was dissolved in chloroform and then evaporated under nitrogen. Chloroform was further removed by placement under low vacuum in a speedvac Savant concentrator (Holbrook, NY). The clodronate solution was prepared by dissolving 1.2 g of dichloromethylene diphosphonic acid (Sigma) in 5 ml of sterile PBS. The clodronate solution (5 ml) was added to the liposome preparation and mixed thoroughly. The solution was sonicated and centrifuged at 10,000 g for 1 h at 4°C. The liposome pellet was removed, resuspended in 5 ml of PBS, and centrifuged at 10,000 g for 1 h at 4°C. Liposomes were removed, resuspended in 5 ml of PBS, and used within 48 h. A single dose of liposomal clodronate was administered via intratracheal injection at 6 wk following FLT.

Bioluminescence imaging.

Mice were anesthetized and shaved over the chest and abdomen before imaging was performed. Luciferin (1 mg/mouse in 200 μl of isotonic saline) was administered by intraperitoneal injection, and mice were imaged with an intensified charge-coupled device camera (model no. C2400-32; Hamamatsu). For the duration of photon counting, mice were placed inside a light-tight box. Light emission from the mouse was detected as photon counts by using the intensified charge-coupled device camera and customized image processing hardware and software (Hamamatsu). A digital false-color photon emission image of the mouse was generated, and photons were counted over a standard area corresponding to the region of the chest overlying the mid-lung zone.

Histology and neutrophil counting.

To collect lung tissue, mice were perfused with saline and lungs were inflated with 1 ml of 10% neutral-buffered formalin. After paraffin embedding, 5-μm sections were cut and placed on charged slides, and standard hematoxylin and eosin (H&E) staining was performed. For each slide, neutrophils were counted in a blinded fashion on 10 sequential, nonoverlapping high-power fields (magnification ×400) of lung parenchyma beginning at the periphery of the section. Three separate H&E-stained sections were evaluated per mouse, and the mean number of neutrophils per high-power field was reported.

Bone marrow-derived macrophages.

Bone marrow-derived macrophages (BMDM) were cultured as follows: mice were euthanized by CO₂ inhalation, and femurs were isolated by surgical resection. The bone marrow was collected by flushing the femurs with medium [DMEM, 10% L929 cell conditioned medium, 10% fetal bovine serum (FBS), and penicillin/streptomycin] using a 27-gauge needle and syringe. After a single-cell suspension was achieved by repeated pipetting, the cells were transferred to a 150-mm plate and placed at 37°C. After 6 days, the medium was removed and BMDM cells were washed once with PBS. The cells were lifted by addition of cold PBS with 5 mM EDTA for 15 min at 4°C. Cells were replated (DMEM, 10% FBS, and penicillin/streptomycin), and experiments were performed starting on day 7 after culture.

A recombinant adenovirus encoding a NF-κB-dependent luciferase reporter (35) was used to detect NF-κB activation in BMDM. For these studies, BMDM were infected with adenoviral vectors at a multiplicity of infection of 300:1. Forty-eight hours later, medium was removed, cells were washed twice with PBS, and new medium was replaced before treatment with LPS.

Coculture.

A549 cells were obtained from ATCC and maintained in DMEM supplemented with 10% FBS and 1% l-glutamine. Coculture experiments were performed using inserts that allow for cell-cell communication via the culture medium. BMDM from wild-type (WT), IκBα^−/+, and p50^−/− mice were cultured as described above. A549 cells were stably transfected with NGL NF-κB reporter plasmid (15). The experiments were performed as follows: day 1, bone marrow was harvested and placed into culture; day 5, A549 cells were plated into bottom wells of coculture plates; day 6, BMDM were plated into upper wells of coculture plates; day 7, 0.2 μg/ml LPS was added to each coculture well, cells were collected at the 4-h time point, and luciferase activity was detected by assay; day 8, cells were collected at the 24-h time point and luciferase detected; and day 9, cells were collected at the 48-h time point and luciferase detected. In some experiments, neutralizing antibodies to IL-1β (1 μg/ml) and TNF-α (1 μg/ml) (both from R&D Systems, Minneapolis, MN) were added to the culture medium along with LPS.

Extraction of nuclear proteins and Western blotting for RelA.

Nuclear proteins were extracted from cells as previously described (6). Protein concentrations in nuclear extracts were determined using the Bradford assay. For Western blot analysis, 25 μg of nuclear protein were separated on a 10% acrylamide gel, transferred to polyvinylidene difluoride membrane, and immunodetected. Anti-RelA antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Measurement of luciferase activity.

Luciferase activity was measured in cells by adding 100 μl of freshly reconstituted luciferase assay buffer to 20 μl of cell lysate in reporter lysis buffer (Promega, Madison, WI). Luciferase activity was expressed as relative light units normalized for protein content, which was measured by Bradford assay.

Chemokine ELISAs.

Macrophage inflammatory protein (MIP)-2, keratinocyte-derived chemokine (KC), and IL-8 were measured using specific ELISA kits according to the manufacturer's instructions (R&D Systems).

Statistical analysis.

Statistical analyses were performed with GraphPad InStat software, version 3.01 for Windows (GraphPad), using an unpaired t-test and unpaired ANOVA. Values of P < 0.05 were considered significant.

RESULTS

Although macrophages are known to be critical for initiation of the lung inflammatory response to gram-negative bacterial LPS and other inflammatory stimuli, we investigated the importance of macrophages in limiting lung inflammatory responses. LPS-induced lung inflammation requires activation of the NF-κB pathway in several different cell types (8), and normal termination of NF-κB signaling in activated cells involves resynthesis of IκBα and new synthesis of the p50 subunit (2). Therefore, we undertook studies to determine whether deficiency of these NF-κB pathway components (IκBα and p50) in bone marrow-derived cells (including macrophages) would affect the duration and intensity of LPS-induced lung inflammation.

To examine the impact of NF-κB activity in myeloid cells on NF-κB activation in nonhematopoietic cells and development of neutrophilic lung inflammation, we generated bone marrow chimeric mice by transplanting stem cells from fetal liver obtained from WT, IκBα^+/−, IκBα^−/−, or p50^−/− embryos to lethally irradiated NF-κB reporter (HLL) mice. We chose HLL mice as recipients because these mice enabled us to monitor recipient NF-κB activity using bioluminescence imaging to detect luciferase activity. After FLT, we treated chimeric mice with an intratracheal injection of liposomal clodronate at 6 wk to eliminate resident alveolar macrophages and ensure repopulation with donor-derived macrophages as previously described (14). At 10 wk after FLT, chimeric mice and untransplanted HLL mice were imaged for baseline NF-κB activation (using luciferase activity as readout) following injection of luciferin. Mice then received a single intraperitoneal injection of LPS (3 μg/g) and were analyzed for NF-κB activation by bioluminescence imaging at 4, 8, 24, and 48 h. Because the chimeric animals did not carry the NF-κB reporter in the donor myeloid cells, the detected luciferase activity was derived from nonhematopoietic cells. As shown in Figs. 1 and 2A, minimal bioluminescence over the thorax (indicative of lung luciferase activity) was identified in all chimeric mice at baseline. After LPS treatment of WT bone marrow chimeras, a transient increase in bioluminescence (indicating NF-κB-dependent luciferase production) was identified at 4 and 8 h, returning to baseline by 24–48 h. Bioluminescence measurements were similar between untransplanted HLL mice and WT chimeras, suggesting that the majority of LPS-induced NF-κB activation occurred in nonhematopoietic cells. In marked contrast, bone marrow chimeras in which myeloid cells contained mutations in IκBα or p50 had continued elevation of luciferase activity at 24 and 48 h post-LPS treatment. At 24 h after LPS treatment, IκBα^−/− bone marrow chimeras showed the greatest activation of NF-κB, followed by p50^−/− bone marrow chimeras and IκBα^+/− chimeras. All IκBα^−/− bone marrow chimeras died between 24 and 48 h after LPS treatment, and 70% of p50^−/− bone marrow chimeras died between 24 and 48 h after intraperitoneal LPS injection. IκBα^+/− bone marrow chimeras had low mortality (<10%), and bioluminescence detected over the thorax returned to baseline by day 6 post-LPS (data not shown). No mortality was observed in untransplanted HLL mice or WT bone marrow chimeras following LPS treatment. Together, these results indicate that the inability to synthesize inhibitory members of the NF-κB family specifically in cells of hematopoietic origin results in generalized prolonged and increased inflammatory signaling in the lung microenvironment. Interestingly, despite the systemic nature of the inflammatory stimulus, the majority of NF-κB activity identified in the IκBα and p50 mutant bone marrow chimeras was localized over the thorax (i.e., lungs).

Fig. 1.

Increased NF-κB activity in lipopolysaccharide (LPS)-treated bone marrow chimeras with IκBα- or p50-deficient myeloid cells. Bioluminescent detection of NF-κB activity in bone marrow chimeras with wild-type (WT), IκBα^+/−, IκBα^−/−, or p50^−/− donor cells into NF-κB reporter transgenic (HLL) mice was performed. After a single intraperitoneal (IP) injection of LPS (3 μg/g), bioluminescence was measured at baseline (BL) and 8, 24, and 48 h following luciferin injection. Images are representative of ≥10 mice per group.

Fig. 2.

NF-κB activity is consistent with neutrophilic lung inflammation in bone marrow chimeras after LPS treatment. A: photons were emitted over the thorax in a group of bone marrow chimeras at 4, 8, 24, and 48 h after IP injection of LPS. In addition to the chimeric mice, untransplanted HLL recipient mice treated similarly with LPS were included. B: quantification of neutrophils in hematoxylin- and eosin-stained lung tissue sections. Neutrophils were counted in 10 nonoverlapping fields per slide. A total of 3 slides per mouse and 3 mice for each time point per experimental group were counted. Results are means ± SE. *P < 0.05 compared with WT chimeras and untransplanted HLL mice at the same time point. HPF, high-power field.

We harvested mice at baseline and 4, 8, 24, and 48 h after intraperitoneal LPS injection for identification of neutrophils in the lung by morphometric evaluation of sections of lung parenchyma. As shown in Fig. 2B, untransplanted HLL mice and WT bone marrow chimeras showed neutrophil influx into the lungs peaking at 8 h and returning to baseline by 48 h. None of these NF-κB mutant bone marrow chimeras exhibited neutrophilic inflammation in the lungs in the absence of LPS; however, after LPS treatment, these mice had prolonged neutrophilic inflammation that mirrored the prolonged and exaggerated NF-κB activation shown in Fig. 2A. On histological evaluation, IκBα^−/− and p50^−/− bone marrow chimeras showed evidence of parenchymal distortion with edema and interstitial thickening at 24 h post-LPS, which was minimal in WT bone marrow chimeras and untransplanted HLL mice (data not shown). In summary, bone marrow chimera experiments indicate that dysregulated NF-κB signaling in cells of hematopoietic origin results in increased neutrophilic inflammation, NF-κB activation in other cell types, histological evidence of lung injury, and high mortality (in IκBα^−/− and p50^−/− chimeras). These findings show that the NF-κB pathway plays an important role in cells of hematopoietic origin to regulate the degree and duration of lung inflammation in response to systemic LPS.

To directly study the impact of NF-κB dysregulation on macrophage phenotype in response to LPS, we generated BMDM from WT, IκBα^+/−, IκBα^−/−, or p50^−/− mice. Cells from WT (IκBα^+/+), IκBα^+/−, and IκBα^−/− mice were infected with a recombinant adenovirus construct that encodes a NF-κB-luciferase reporter construct and were then treated with LPS (0.2 μg/ml) for up to 24 h before measurement of luciferase activity. As shown in Fig. 3A, LPS treatment of BMDM induced differential NF-κB-mediated transcription in BMDM that peaked by 4 h after LPS treatment. Both IκBα^+/− and IκBα^−/− BMDM showed increased luciferase activity at 4 h after LPS compared with WT controls. NF-κB transcriptional activity decreased to near basal level by 24 h after LPS treatment in the WT and IκBα^+/− cells, but IκBα^−/− BMDM still retained active NF-κB at the 24-h time point. To study p50-deficient cells, we cross-mated p50-deficient mice with HLL NF-κB reporter mice to generate p50^−/−/HLL mice. We then cultured BMDM from p50^−/−/HLL mice and treated them in vitro with LPS as described above. In these experiments, NF-κB-dependent luciferase expression was significantly increased at 4 and 24 h in the absence of p50 compared with p50^+/+ HLL control BMDM (Fig. 3B). On the basis of results of the NF-κB reporter assays, we performed Western blot analyses for RelA from nuclear protein extracts obtained from cells at the time of harvest. Consistent with the reporter assays, no differences were identified in nuclear translocation of RelA in untreated BMDM from different genotypes, but nuclear RelA was increased in IκBα^−/− and p50^−/− cells at 4 and 24 h after LPS treatment (Fig. 3C).

Fig. 3.

Increased NF-κB activity in IκBα- and p50-deficient bone marrow-derived macrophages (BMDM) after LPS treatment. A: BMDM from IκBα^−/−, IκBα^+/−, and IκBα^+/+ (WT) mice were infected with adenoviral vectors encoding an NF-κB reporter with a multiplicity of infection of 300. At 48 h after adenoviral infection, cells were treated with LPS (0.2 μg/ml) and luciferase activity was measured in cell lysates and normalized for total protein. Results are reported as relative light units (RLU) and presented as means ± SE; n = 4 per group. *P < 0.05 compared with IκBα^+/+ (WT) BMDM at the same time point. B: p50^−/−/HLL and p50^+/+ (WT)/HLL BMDM were treated with LPS as described in A, and luciferase activity was measured from cell lysates. Results are means ± SE; n = 4 per group. *P < 0.05 compared with p50^+/+ (WT)/HLL at the same time point. C: Western blot for nuclear RelA in BMDM. Results are representative of 3 separate experiments. Equal protein loading was determined by immunodetection of TATA-box binding protein on each gel (not shown).

Since LPS-induced neutrophil recruitment is primarily due to NF-κB-directed production of C-X-C chemokines (7, 36, 38), we measured production of MIP-2 and KC in cell culture media from BMDM after LPS treatment. As shown in Fig. 4A, IκBα^−/− and p50^−/− BMDM generated more MIP-2 than WT BMDM at 4 and 24 h after LPS treatment. For KC, IκBα^−/− BMDM produced a larger amount than WT cells, but p50^−/− BMDM generated no increase in KC production after LPS, suggesting that p50 is required for LPS-induced KC gene transcription (Fig. 4B).

Fig. 4.

Differential chemokine production in IκBα- and p50-deficient BMDM. BMDM were treated with LPS (0.2 μg/ml), culture medium was collected, and the concentrations of macrophage inflammatory protein-2 (MIP-2; A) and keratinocyte-derived chemokine (KC; B) were determined by specific ELISA. Results are means ± SE; n = 4 per group. *P < 0.05 for increase compared with IκBα^+/+ (WT) BMDM at the same time point.

In addition to macrophages and other bone marrow-derived cells, structural cells, particularly lung epithelium, are prominent producers of inflammatory mediators. Our recent studies have shown that NF-κB activation in lung epithelial cells plays an important role in neutrophilic lung inflammation following LPS treatment (15, 34). Since bone marrow chimera studies showed increased NF-κB activity in nonhematopoietic cells after intraperitoneal injection of LPS, we hypothesized that dysregulated NF-κB activation in macrophages leads to increased NF-κB signaling in epithelium. To investigate this idea, we cocultured macrophages on inserts with A549 lung epithelial cells. A549 epithelial cells were chosen because they have characteristics similar to type II alveolar epithelium, are minimally responsive to LPS, and are human in origin. Utilization of A549 cells allowed us to identify with certainty the epithelial origin of human NF-κB-dependent gene products. BMDM from WT, IκBα^−/−, or p50^−/− mice were grown on inserts and placed in coculture with A549 cells stably transfected with a NF-κB reporter construct to allow communication between cells through the production of soluble mediators. The coculture was treated with 0.2 μg/ml LPS, which does not activate NF-κB in A549 cells (data not shown). Cells were harvested for luciferase measurements, and media were saved at 4, 24, and 48 h after LPS. As shown in Fig. 5A, culturing IκBα^−/− or p50^−/− BMDM with A549 cells resulted in increased NF-κB-dependent luciferase production in A549 cells at 24 and 48 h after LPS treatment compared with cocultures with WT BMDM. We also measured IL-8 in media, since this human chemokine is regulated by NF-κB (41) and produced by human, but not murine, cells. Similar to NF-κB activation in A549 cells cocultured with BMDM, production of IL-8 in A549 cells was increased at 24 and 48 h after LPS treatment when cells were cocultured with IκBα^−/− or p50^−/− BMDM (Fig. 5B). Collectively, these data show that dysregulated NF-κB signaling in macrophages increases the responsiveness of these cells to LPS and stimulates other cells in the environment to increase NF-κB activation and chemokine production. As suggested by our experimental data, these interactions between macrophages and epithelial cells may be particularly important for regulating inflammatory signaling at later time points after an inflammatory stimulus.

Fig. 5.

NF-κB activity in macrophages regulates NF-κB activation and chemokine production in lung epithelial cells through production of TNF-α and IL-1β. A: BMDM from WT, IκB-α^−/−, or p50^−/− mice were placed in the top chamber and A549 cells stably transfected with a NF-κB reporter plasmid were grown in the bottom chamber of a coculture apparatus. After treatment of the cells with 0.2 μg/ml LPS for indicated periods, NF-κB activity in A549 cells was measured by luciferase assay. No increase in NF-κB activity was observed after LPS treatment of A549 cells in the absence of cocultured macrophages (data not shown). B: human IL-8 production by A549 cells was measured by ELISA after treatment of cocultures with LPS. Results are means ± SE; n = 3 samples per group. Data are representative of 3 separate experiments. *P < 0.05 compared with coculture with WT BMDM at the same time point. C: experimental design similar to A with the addition of neutralizing antibodies to TNF-α and IL-1β (1 μg/ml each) at the time of LPS treatment. Cells were harvested for luciferase activity assays at 24 h after LPS. Results are means ± SE; n = 6 samples per group in 2 separate experiments. *P < 0.05 compared with coculture with WT BMDM. **P < 0.05 compared with coculture with WT BMDM + TNF-α/IL-1β antibodies.

TNF-α and IL-1β are NF-κB-dependent cytokines produced by macrophages in response to LPS and can activate NF-κB signaling in many cell types, including lung epithelium. Therefore, we wondered whether these mediators were responsible for induction of NF-κB activation in A549 cells in our coculture system. To address the role of TNF-α and IL-1β in activating NF-κB in lung epithelial cells, we added neutralizing antibodies (1 μg/ml each) to cocultures along with LPS and harvested A549 cells containing the NF-κB-luciferase reporter at 24 h. As shown in Fig. 5C, inhibition of TNF-α and IL-1β signaling reduced A549 cell luciferase activity in cocultures with WT BMDM to the level of A549 cells cultured alone. However, this was not the case in cocultures with IκBα^−/− and p50^−/− BMDM, where TNF-α and IL-1β blockade failed to eliminate the increase in A549 luciferase activity. These data indicate that TNF-α and IL-1β are responsible for the induction of NF-κB activity in A549 epithelial cells by cocultured LPS-treated WT macrophages. However, in the case of IκBα and p50 deficiency, macrophages appear to produce additional factors that stimulate NF-κB activation in epithelial cells.

DISCUSSION

Although alveolar macrophages have a well-recognized role in generation of inflammatory responses in the lungs, our studies highlight an important, active role for these cells in limiting and terminating neutrophilic inflammation through expression of inhibitory NF-κB components. Macrophages deficient in inhibitory components of the NF-κB pathway show increased NF-κB transcriptional activity and chemokine production after LPS treatment. These p50- and IκBα-deficient macrophages also induce increased NF-κB signaling and chemokine production in cocultured alveolar epithelial cells, consistent with the idea that products of NF-κB pathway signaling in macrophages regulate NF-κB activation in the surrounding microenvironment. In vivo, deficiency of IκBα or p50 in myeloid cells resulted in excessive and prolonged neutrophilic inflammation and NF-κB activation in nonhematopoietic cells. As a result, IκBα^−/− or p50^−/− bone marrow chimeric mice developed histological evidence of lung injury and high mortality after systemic endotoxemia. Together, these studies indicate that communication and interplay between myeloid and nonmyeloid cells lead to a coordinated NF-κB response that regulates the outcome of lung inflammation.

Germ-line deficiency of IκBα results in perinatal lethality with death occurring at days 7–10 in the presence of widespread inflammation, dermatitis, and excessive granulopoiesis (3, 20). In contrast, mice with a selective deficiency of IκBα in myeloid cells or IκBα^−/− bone marrow chimeras have normal peripheral white blood cell counts and differentials (33), consistent with a lack of spontaneous inflammation observed in our studies. In vitro, cultured IκBα^−/− BMDM showed no increase in basal NF-κB activity, similar to previous studies with embryonic fibroblasts (20). Therefore, in the macrophage population, other inhibitory NF-κB components appear to compensate for the loss of IκBα under basal conditions, and a proinflammatory phenotype is identified only in the presence of an activating stimulus. These studies define an important role for IκBα in postinduction repression of NF-κB activity in macrophages.

In contrast to IκBα-deficient mice, p50 knockout mice develop normally but show defects in lymphocyte function and altered susceptibility to infection (37). In a model of E. coli pneumonia, p50-deficient mice had increased cytokine production and neutrophil recruitment without impacting bacterial clearance (30). Surprisingly, airway delivery of E. coli LPS resulted in reduced neutrophil recruitment despite increased cytokine production (31). In our studies, however, p50^−/− bone marrow chimeras displayed a clear proinflammatory phenotype that was mirrored by the results of experiments using p50^−/− BMDM. Therefore, the preponderance of evidence points to a primary function of the p50 subunit to limit or repress NF-κB signaling, similar to IκBα. Potentially, this could be due to generation of nonsignaling p50 homodimers or repressive effects of the p105 precursor.

In addition to our studies, other investigations of the roles of specific NF-κB pathway components in myeloid cells have uncovered interesting and unexpected results. Bone marrow chimeras deficient in the major NF-κB activating subunit, RelA, have normal neutrophil recruitment to the lungs following airway delivery of LPS (1). This report suggests that other cell types in the lungs provide the crucial signals for neutrophil recruitment. Deficiency of IKKα in myeloid cells increases the systemic inflammatory response to LPS and worsens survival (24). Further experiments have indicated that IKKα limits inflammation by accelerating turnover of RelA and c-Rel and facilitating their removal from gene promoters (24). Deficiency of IKKβ in macrophages also increases susceptibility to endotoxic shock following large doses of E. coli LPS (30 μg/g) (18). In this case, the poor outcome appeared to be due to increased IL-1β release (18). Together with the current report, available evidence suggests that an intact NF-κB pathway response in myeloid cells is required for limiting the degree and duration of the inflammatory response to bacterial LPS.

Another important finding in our studies is the coordinated NF-κB response in myeloid and nonmyeloid cells to LPS treatment. In vitro, BMDM deficient in IκBα or p50 demonstrated increased NF-κB transcription that peaked at 4 h after LPS and returned toward baseline by 24 h. In contrast, cocultured epithelial cells had sustained NF-κB activation in the presence of IκBα^−/− or p50^−/− BMDM to 48 h, whereas epithelial cells cocultured with WT BMDM returned toward baseline at this time point. This finding has implications for our in vivo studies, where NF-κB activation in nonhematopoietic cells continued to increase through 24–48 h. In recent studies, we have shown that 1) NF-κB activation occurs in a variety of cell types in the lungs in response to systemic LPS, 2) the duration of NF-κB signaling is associated with the development of lung injury following LPS, and 3) airway epithelial cells have a major role in determining whether LPS-induced inflammation resolves or progresses to lung injury (13, 15). Therefore, it appears likely that inability to limit NF-κB signaling in NF-κB mutant myeloid cells propagates NF-κB signaling in epithelial cells and other parenchymal cell types through production of soluble mediators, and this enhanced signaling in epithelial cells exacerbates neutrophilic lung inflammation. On the basis of our coculture studies, it is clear that TNF-α and IL-1β participate in the coordinated NF-κB response between macrophages and epithelial cells. These cytokines are produced by macrophages and activate NF-κB through their cognate receptors on epithelial and other parenchymal cells. Interestingly, neutralization of TNF-α and IL-1β eliminated the induction of NF-κB activity in A549 cells cocultured with WT macrophages but was only partially effective when added to cocultures with IκBα^−/− or p50^−/− macrophages. These findings indicate that loss of inhibitory NF-κB components in macrophages may enhance production of additional soluble factors capable of activating NF-κB in neighboring cells.

Although our studies focused on regulation of inflammation by myeloid cells, cross talk between myeloid and parenchymal cells can occur in both directions. A recent study using cocultures of airway epithelial cells and BMDM found that activating NF-κB in epithelial cells by expression of a constitutively active form of IKKβ resulted in NF-κB activation and cyclooxygenase-2 (COX-2) gene expression in cocultured macrophages (27). These findings show the integrated nature of NF-κB signaling in the lungs.

In summary, our results indicate that alveolar macrophages communicate with other lung cell types and that the NF-κB pathway in macrophages plays a determining factor in severity and duration of lung inflammation. Our results also highlight an important interaction between myeloid and nonmyeloid cells in regulating inflammatory responses in the lungs. Hopefully, improved understanding of mechanisms that regulate lung inflammation and injury will lead to novel therapeutic approaches to improve outcome in patients with acute lung injury.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL61419 and HL66196, the U.S. Department of Veterans Affairs, Vanderbilt Ingram Cancer Center, and Department of Defense Breast Cancer Program Grant WX1XWH-04-1-0456.