Rationale: Clearance of apoptotic cells is crucial to the resolution of inflammation and development of fibrosis, but the process is not well understood in normal or diseased human lungs.
Objectives: To determine phagocytosis of apoptotic cells by primary human alveolar macrophages and whether defects in uptake of apoptotic cells are associated with decreases in antiinflammatory/antifibrotic mediators.
Methods: Human bronchoalveolar lavage macrophages (AMφs) from normal control subjects and subjects with mild-moderate or severe asthma were examined in vitro for phagocytosis of apoptotic human T-cell line Jurkats and secretion of inflammatory mediators.
Measurements and Main Results: AMφs from normal subjects and patients with mild-moderate asthma were able to phagocytose apoptotic cells in response to LPS, resulting in an induction of the antifibrotic and/or antiinflammatory eicosanoids, prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (HETE). In contrast, AMφs from patients with severe asthma had defective LPS-stimulated uptake of apoptotic cells, with associated failure to induce PGE2 and 15-HETE. In addition, LPS-stimulated basal levels of tumor necrosis factor α and granulocyte-macrophage colony–stimulating factor were reduced in all patients with asthma, whereas PGE2 and 15-HETE were reduced only in patients with severe asthma. Dexamethasone enhanced specific uptake of apoptotic cells in all subjects, while suppressing inflammatory mediator secretion.
Conclusions: A decrease in AMφs LPS-responsiveness in severe asthma is manifested by defective apoptotic cell uptake and reduces secretion of inflammatory mediators. This may contribute to the chronicity of inflammation and remodeling in lungs of patients with asthma.
Clearance of apoptotic cells by phagocytes is crucial in maintaining tissue homeostasis. This process prevents dying cells from releasing proinflammatory contents into the environment. In animal and in vitro studies, apoptotic cell clearance induces secretion of antiinflammatory mediators, such as transforming growth factor β1 (TGF-β1) and prostaglandin E2 (PGE2), with associated suppression of proinflammatory cytokines, chemokines, and eicosanoids (1–3). The physiologic significance of this phenomenon has been confirmed in a murine model in which in vivo clearance of apoptotic cells accelerates the resolution of LPS-induced lung inflammation (2), mediated partly by induction of TGF-β1. Both unstimulated murine (4–6) and human monocyte–derived macrophages (1) have been demonstrated to phagocytose apoptotic cells, which is markedly enhanced after activation with LPS, zymosan (1, 2), glucocorticoids (7), and lipoxins (8).
Regulation of apoptotic cell clearance in human lungs, either in the normal or inflamed/diseased state, remains poorly understood, as most previous studies have been limited to animal models, cell lines, and human monocyte–derived macrophages. We sought to examine clearance in human alveolar macrophages from normal lungs and from lungs with asthma (9). Apoptotic cell uptake was examined in freshly harvested human bronchoalveolar lavage (BAL) macrophages (AMφs) from normal lungs and lungs of patients with mild–moderate and severe asthma. We hypothesized that defects in apoptotic cell uptake by AMφs from asthmatic lungs could contribute to the chronic inflammation seen in this disease.
Human T-lymphocyte Jurkat cells (American Type Tissue Culture Collection, Manassas, VA) were cultured in Roswell Park Memorial Institute 1640 medium (10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 37°C, 5% CO2). Apoptosis of Jurkats was induced by ultraviolet irradiation (245 nm, 10 min) and used at 60–80% apoptosis (incubated for 2–4 h, assessed morphologically by light microscopy). Apoptotic Jurkats were opsonized with mouse antihuman CD45 IgG1 (Pharmingen, San Diego, CA) (2).
See online supplement for details.
Patients with severe asthma were referred to the National Jewish Medical and Research Center for severe oral corticosteroid–dependent asthma. These subjects had frequent hospitalizations and/or emergency room visits, evidence for ongoing severe airflow limitation (FEV1 < 70% predicted), and oral or high-dose inhaled corticosteroid use (10). Patients with moderate asthma had FEV1 less than 80% predicted on 400–1,000 μg of inhaled corticosteroids and β-agonists. Patients with mild asthma had an FEV1 greater than 80% predicted on β-agonists alone. Normal control subjects had normal pulmonary function, no bronchodilator response, and no history of respiratory illness. Some patients with severe asthma were treated in vivo with a high-dose corticosteroid burst (prednisone equivalent of at least 40 mg/d × 7–10 d), and BAL was again performed.
BAL cells were isolated and cytospins made (11). AMφs were isolated by adhesion (60 min) and cultured in serum-free X-vivo 10 + gentamycin media (37°C, 5% CO2) for all experiments (see online supplement for details).
Suspended, unfixed AMφs were stained with trypan blue (0.08%, 1 min) and examined with light microscopy. DNA fragmentation of apoptotic cells were detected in cytospins using the enzyme terminal deoxynucleotidyl transferase with the DeadEnd colorimetric transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) system (Promega, Madison, WI; see online supplement for details).
The AMφs (5 × 104 AMφs/12-mm glass coverslip) were stimulated with 10 μg/ml LPS for 2–8 h, and/or dexamethasone (Dex, 0.1–50.0 μM) for 1–6 h. Because no difference was found between addition of Dex before, during, or after introduction of LPS, Dex was added after LPS for all subsequent experiments. Apoptotic Jurkats were added to AMφ cultures (ratio of 20:1 in 250 μl media) for 60 min. Phagocytic bodies were assessed by light microscopy in a blinded fashion; phagocytosis was quantified by the phagocytic index (PI, %), which represents the number of phagocytic bodies per 200 AMφs × 100 (12). Controls included cell-free media, viable Jurkats, and opsonized apoptotic Jurkats.
A total of 0.3–0.4 × 106 AMφs/well were stimulated with LPS (10 μg/ml, 8 h), followed by Dex (1 μM, 1 h). Apoptotic Jurkats were incubated with AMφs (ratio of 10:1 in 250 μl medium) for 2 h, and then replaced with 500 μl of fresh media. The AMφ cultures were incubated for 18 h, and the supernatants were assayed for cytokine and eicosanoid concentrations using ELISAs. Similar controls were performed as described above.
Human tumor necrosis factor α (hTNF-α), human granulocyte-macrophage colony–stimulating factor (hGM-CSF), hPGE2, human 15-hydroxyeicosatetraenoic acid (h15-HETE), and hTGF-β1 and hTGF-β2 were measured from the 18-h supernatants of cultured AMφs (see online supplement for details).
Analysis of variance with Student's t test and Dunnett's method was performed using JMP software (SAS Institute, Cary, NC)
The profiles of normal control subjects and subjects with asthma included in the retrospective review of BAL cytospins (Figure 1)are presented in Table 1
BAL Cytospin Demographics
|Age, yr||37.21 ± 2.96||35.64 ± 2.23||33.88 ± 2.36||NS|
|BAL cell count × 104/ml||15.69 ± 2.79||14.88 ± 0.96||13.34 ± 1.96||NS|
|Monocyte/macrophage, %||84.28 ± 2.88||87.21 ± 1.85||82.82 ± 2.78||NS|
|Lymphocytes, %||11.36 ± 2.78||8.33 ± 1.20||8.26 ± 2.16||NS|
|Neutrophils, %||1.28 ± 0.44||0.74 ± 0.08||2.31 ± 0.67||NS|
|FEV1||3.59 ± 0.19||2.97 ± 0.18*||1.75 ± 0.17*||< 0.0001|
|FVC||4.41 ± 0.27||4.32 ± 0.25||2.74 ± 0.21*||< 0.0001|
|Change after bronchodilator, %||4.02 ± 0.53||15.12 ± 2.41*||31.65 ± 6.10*||< 0.0001|
|FEV1/FVC ratio||0.82 ± 0.02||0.69 ± 0.02*||0.63 ± 0.03||< 0.0001|
|Corticosteroid use (inhaled + oral)||No||16 No (mild)/9 yes (moderate)||Yes|
|Oral corticosteroid, %||0||0||83|
|Daily predisone equivalent dose, mg||NA||NA||36.89 ± 8.34|
|Age, yr||30.68 ± 2.09||30.917 ± 3.26||31.75 ± 2.65||NS|
|BAL cell count × 104/ml||23.10 ± 4.20||15.6125 ± 2.24||23.90 ± 3.12||NS|
|Monocyte/macrophage, %||86.14 ± 2.80||91.76 ± 1.37||77.72 ± 2.74*||< 0.012|
|Lymphocytes, %||10.33 ± 2.70||5.18 ± 0.103||16.33 ± 2.81||NS|
|Neutrophils, %||1.16 ± 0.31||0.51 ± 0.10||3.38 ± 1.35||NS|
|FEV1||3.92 ± 0.23||3.11 ± 0.31*||2.04 ± 0.15*||< 0.03|
|FVC||4.63 ± 0.26||4.51 ± 0.44||3.43 ± 0.25*||< 0.002|
|Change after bronchodilator, %||3.20 ± 1.046||15.9 ± 3.27*||27.44 ± 4.52*||< 0.007|
|FEV1/FVC ratio||0.85 ± 0.01||0.69 ± 0.04*||0.64 ± 0.03*||< 0.001|
|Corticosteroid use (inhaled + oral)||No||7 No (mild)/5 yes (moderate)||Yes|
|Oral corticosteroid, %||0||0||100|
|Daily predisone equivalent dose, mg||NA||NA||23.03 ± 0.03|
In Table 1, normal control subjects and patients with asthma were similar with regard to age, sex ratio, and BAL total cell counts and differentials. In the second population represented in Table 2, normal control subjects and patients with asthma were similar with regard to age, sex, and BAL total cell counts. The combined mild and moderate asthma group was smaller in size, more likely to be female (8/12), and had less total cells. Patients with severe asthma had a significantly lower percentage of macrophages, and trends for both higher lymphocyte and neutrophil percentages.
Indirect evidence of human AMφ phagocytosis was obtained by retrospectively comparing the phagocytic bodies present in BAL cytospin AMφs obtained from normal subjects and patients with mild–moderate and severe asthma. AMφs from normal subjects and patients with mild–moderate asthma had similar numbers of phagocytic bodies (quantified by PIs; Figure 1). In contrast, AMφs from patients with severe asthma had a reduced PI compared with all other subjects. Twelve patients with severe asthma were treated with a high-dose oral corticosteroid burst (prednisone equivalent of at least 40 mg/d × 7–10 d), followed by a BAL. The PI was significantly increased in the postburst cytospin AMφs compared with preburst.
To begin to address these discrepancies between AMφs from normal subjects and those from patients with severe asthma in vivo, the phagocytic activities of normal AMφs were assessed in vitro. Freshly harvested AMφs from normal control subjects were cultured and exposed to cell-free media, live human T-lymphocyte Jurkat cells (ViableJ), apoptotic Jurkats (ApoJ), or opsonized apoptotic Jurkats (OpsJ) for 60 min. The phagocytic efficiency (quantified by the PI) was increased in AMφs exposed to ApoJ compared with cells exposed to cell-free media, ViableJ, and OpsJ (Figure 2). Thus, unstimulated normal human AMφs are able to phagocytose apoptotic cells, but not viable or opsonized cells.
The regulation of apoptotic cell uptake by LPS and the corticosteroid Dex in normal control AMφs was then examined. Normal control AMφs were stimulated with LPS and/or Dex, followed by exposure to cell-free media, ViableJ, ApoJ and OpsJ. LPS increased the phagocytosis of both ApoJ and OpsJ, suggesting a global activation of the AMφs (Figure 2B). In contrast, preexposure to Dex increased the uptake of only ApoJ, and had no effect on ViableJ or OpsJ (Figure 2C). The LPS + Dex combination had no additional effect over either stimulus alone (Figure 2D). LPS induction of uptake of ApoJ increased linearly with increased stimulation time, and 8 h of LPS stimulation was subsequently chosen for the remaining experiments to minimize AMφs ex vivo time (Figure 3A). Maximal effects of Dex on uptake of ApoJ were observed within 30 min and at 1 μM concentration (Figures 3B and 3C).
The reduced in vivo PI in BAL cytospin AMφs from patients with severe asthma (Figure 1) may have resulted from either decreased or increased phagocytosis efficiency, as well as reduced availability of phagocytic targets. Phagocytosis by AMφs from patients with mild–moderate and severe asthma was thus examined in vitro using freshly isolated AMφs. AMφs from normal control subjects and patients with asthma were cultured and exposed to Jurkats as described above. In patients with mild–moderate asthma, unstimulated AMφs could phagocytose ApoJ (Figure 4)as in normal AMφs. LPS further enhanced uptake of both ApoJ and OpsJ, whereas Dex enhanced specific uptake of ApoJ. Thus, phagocytosis of Jurkats cells by AMφs from patients with mild–moderate asthma was similar to that in AMφs from normal control subjects.Figures 4 and 5B), yet LPS was able to enhance phagocytosis of OpsJ. Conversely, the upregulation of uptake of ApoJ by Dex in AMφs from patients with severe asthma was similar to that of the other subject groups (Figure 5C), consistent with the increased PI found in vivo in the postcorticosteroid BAL cytospin AMφs. Thus, AMφs from patients with severe asthma appear to have a defect in basal and LPS-stimulated uptake of apoptotic cells.
Because the defect in uptake of apoptotic cells by AMφs from patients with severe asthma was linked to LPS responsiveness, the impact of LPS on inflammatory mediator secretion was also examined. AMφs from normal subjects and those with asthma were stimulated with LPS or Dex. Protein concentrations of TNF-α, GM-CSF, transforming growth factor β1 (TGF-β1), TGF-β2, PGE2, and 15-HETE were measured in the supernatants at 18 h. Mediator levels were minimally detected in unstimulated and Dex-stimulated AMφs from all groups (data not shown). LPS stimulation significantly increased the secretion of TNF-α, GM-CSF, PGE2, and 15-HETE in all subject groups (Figure 6). However, the magnitude of the LPS induction of TNF-α and GM-CSF was significantly lower in all AMφs from patients with asthma compared with those from normal subjects. PGE2 and 15-HETE levels were significantly reduced in AMφs from patients with severe asthma compared with normal control subjects and patients with mild–moderate asthma. In contrast to other mediators, supernatant TGF-β1 and β2 proteins were minimally detectable in all subjects even after LPS stimulation (ELISAs for TGF-β1 and β2 had lower detection limits of 10 pg/ml).
To determine the functional consequence of defective uptake of apoptotic cells by AMφs from patients with severe asthma, mediator secretion was measured after exposure to ApoJ in the presence and absence of LPS. In unstimulated AMφs from all subject groups, ApoJ had no effect on baseline mediator levels (data not shown). After LPS stimulation, the addition of ApoJ to AMφs from normal subjects and patients with mild–moderate asthma further increased PGE2 and 15-HETE secretions over levels from LPS stimulation alone (Figure 7). There was no effect on TNF-α, GM-CSF, TGF-β1, or TGF-β2 secretion. In contrast, the addition of ApoJ to AMφs from patients with severe asthma (after LPS stimulation) had no effect on PGE2 or 15-HETE secretions. The lack of effect of ApoJ on TNF-α, GM-CSF, TGF-β1, or TGF-β2 secretion in patients with severe asthma was similar to control subjects. Exposure to ViableJ and OpsJ had no effect on mediator secretion.
To ensure that the above findings did not result from differences in AMφ viability between subject groups, the viability of the harvested AMφs was assessed by trypan blue dye exclusion and DNA fragmentation. Low levels of positively stained trypan blue dye were demonstrated in all subject groups, consistent with high viability (Figure 8A). An earlier marker of apoptosis, chromosomal DNA fragmentation, was detected by the TUNEL assay. Low levels of positively stained cells by TUNEL reaction (DNA fragmentation) were seen in both AMφs from normal control subjects and those from patients with severe asthma (Figure 8B).
In this study, differences in uptake of apoptotic cells and secretion of inflammatory mediators were observed between primary human AMφs from normal control subjects and patients with asthma. Phagocytosis of apoptotic cells by AMφs from normal control subjects and patients with milder asthma was associated with an induction of the antiinflammatory and/or antifibrotic eicosanoids, 15-HETE and PGE2. In contrast, AMφs from patients with severe asthma had defective basal and LPS-mediated uptake of apoptotic cells, with associated failure to induce PGE2 and 15-HETE.
Although unstimulated normal human AMφs inefficiently phagocytose apoptotic cells (as reported for other cell types) (4–6), this process could be enhanced by preactivation with LPS or Dex (1, 7, 13). LPS-mediated phagocytosis of apoptotic cells induced secretion of the antiinflammatory and/or antifibrotic eicosanoids, PGE2 and 15-HETE (Figure 7). LPS stimulation resulted in a global activation of the AMφs, as it also stimulated secretion of inflammatory mediators and phagocytosis of opsonized cells. In contrast, corticosteroid-mediated phagocytosis was specific to apoptotic cells, with associated suppression of inflammatory mediators.
In this first study of apoptotic cell uptake in asthma, AMφs from patients with severe asthma, but not from patients with mild–moderate asthma, appeared to have decreased in vivo phagocytic bodies, which then increased after an in vivo corticosteroid burst (Figure 1). Although this in vivo finding may have resulted from decreased phagocytosis, it can also be seen in cells with increased phagosome degradation efficiency or reduced availability of phagocytic targets. We agree that this is only a measure of intracellular phagocytic bodies. However, using phagocytosis assays, we proceeded to show that, in vitro, AMφs from patients with severe asthma demonstrated defective phagocytosis of apoptotic cells in both unstimulated and LPS-stimulated states that was restored by corticosteroid treatment (Figures 4 and 5). This suggests that similar defects in clearance may exist in vivo (Figure 1).
The LPS-mediated defect was specific for apoptotic cell uptake, as phagocytosis of cells via other mechanisms was intact (e.g., opsonized apoptotic cells via Fcγ receptor ligation). However, a decreased LPS response was also observed in the finding that LPS stimulated lower levels of inflammatory mediators in all patients with asthma compared with normal control subjects, and, in patients with severe asthma, the decreased secretion of PGE2 and 15-HETE. Finally, the LPS-related defective apoptotic cell uptake was associated with failure to induce PGE2 and 15-HETE in AMφs from patients with severe asthma.
This suppressed LPS inflammatory mediator response was also observed by several authors. Chanez and colleagues demonstrated that LPS stimulated secretion of interleukin 1, TNF-α, and interleukin 6 in AMφs from patients with asthma was decreased when compared with nonatopic normal control subjects (14). Data from one study showing increased LPS-stimulated GM-CSF and TNF-α levels in AMφs from patients with asthma could not be compared with our data because the control group consisted mostly of atopic and smoking subjects, which may influence AMφs activation state (15). The decreased production of PGE2 was consistent with findings in airway smooth muscle, suggesting decreased PGE2 and cyclooxygenase-2 expression in asthma (16). Defective phagocytosis of apoptotic cells was also demonstrated in unstimulated AMφs from patients with chronic obstructive pulmonary disease (17). This may suggest that defective phagocytosis of apoptotic cells is a common feature of chronic lung diseases.
Some of our findings were surprising. The previously described increase in 15-HETE levels in severe asthma lavage fluid and tissue (18, 19) may suggest that other cell types (epithelial cells and eosinophils) are the primary source in the lung tissue. Surprisingly, in contrast to other models (1, 13, 20–23), TGF-β1 and TGF-β2 were only minimally secreted by human AMφs and unaffected by apoptotic cell clearance, further supporting the necessity of using primary human cells in research. In fact, actual TGF-β protein secretion has been low in other studies using primary normal human AMφs (24–26), in spite of being detectable by immunohistochemistry and polymerase chain reaction (27, 28). One report of increased TGF-β1 secretion in AMφs from patients with asthma (24) was confounded by the presence of fetal bovine serum, which may have influenced the measured TGF-β1 level. Finally, previously reported defective Fc receptor–mediated phagocytosis (29, 30) in asthma was not observed in this study, which may be due to different patient and cell population (sputum/peripheral blood macrophage) and activation state of the obtained cells (inhaled LPS was used to recruit leukocytes).
The mechanism behind the defective apoptotic cell uptake with LPS in AMφs from patients with severe asthma is most likely complex, involving both intrinsic and extrinsic factors. An intrinsic defect in apoptotic cell uptake mechanisms may be present in AMφs from patients with asthma, but the observation that LPS-stimulated mediator secretion was suppressed and apoptotic cells could be phagocytosed in response to corticosteroids both suggested defects in LPS signaling pathways. Viabilities of the AMφs were similar between all subject groups (Figure 8), so other mechanisms are required to explain the defective apoptotic cell phagocytosis and LPS signaling.
Although an effect of chronic systemic corticosteroid use to suppress the LPS response (Figure 6) is difficult to exclude, the low levels of TNF-α and GM-CSF after LPS stimulation in non–steroid-treated patients with mild asthma suggest that other factors may contribute to the suppressed LPS response. Interestingly, an acute treatment with corticosteroids enhanced uptake of apoptotic cells, but chronic steroid use was insufficient to restore the defective phagocytosis found exclusively in AMφs from patients with severe asthma. Whether chronic versus acute steroid use could have opposite effects can only be answered by direct comparison of chronic versus acute steroid exposure in normal subjects. These studies are difficult to execute. Other environmental and intrinsic factors involved in suppressed LPS response are currently being investigated in our laboratory.
In conclusion, this study expands previous studies in human monocyte–derived macrophages, murine models, and cell lines to human airway cells in the inflammatory disease of asthma. First, in normal AMφs, classically opposing inflammatory (LPS) and antiinflammatory (Dex) mediators were both shown to upregulate apoptotic cell uptake, enabling resolution of inflammation. One appears to work through specific secretion of antiinflammatory/antifibrotic eicosanoids, whereas the other is associated with more generalized suppression of inflammatory mediators. Corticosteroid induction of apoptotic cell clearance in vivo may be yet another mechanism for the therapeutic effect of corticosteroids. Finally, AMφs from patients with asthma may have a suppressed response to LPS, manifested by suppressed secretions of inflammatory mediators in all patients with asthma and the defective apoptotic cell uptake seen in patients with severe asthma. The functional consequence of these suppressed responses in AMφs from patients with asthma may be disadvantageous to resolution of inflammation (31, 32). By failing to remove dying cells, with the adjunctive loss of 15-HETE and PGE2 responses, AMφs could potentially contribute to chronic inflammation, abnormal remodeling, and bronchospasm in asthma.
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